Treating an atypical protein kinase c enzyme abnormality

ABSTRACT

Provided herein are compounds, compositions, pharmaceutical formulations, methods of treating and methods of using aPKC inhibitors for treating and/or preventing of aPKC abnormalities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 61/859,875 filed on Jul. 30, 2013, having the title “Compositionsand Methods for Treating an Atypical Protein Kinase C EnzymeAbnormality,” the disclosure of which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under Contract No.7RO1DK 065969-09 awarded by the National Institutes of Health and a VAMerit Review Grant awarded by the Department of Veterans Affairs. TheU.S. Government has certain rights in this invention.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form asan ASCII.txt file entitled Sequence Listing 292103-2270.txt, created onJul. 30, 2014, and having a size of 2,954 bytes. The content of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND

Obesity is an epidemic that has become the leading preventable cause ofdeath worldwide. Authorities internationally view obesity as one of themost serious public health problems of the 21^(st) century. In 2013, theAmerican Medical Association classified obesity as a disease. Generally,obesity is the condition in which excess body fat has accumulated to theextent that adversely affects morbidity and mortality. In the UnitedStates, obesity is estimated to cause between 112,000 and 365,000 deathsper year and reduces life expectancy by approximately six to sevenyears. Comorbities such as type-2 diabetes and metabolic syndrome arecharacterized by glucose intolerance, high blood pressure, high bloodcholesterol, and high triglyceride levels.

Health complications are either caused directly by obesity or indirectlythrough related mechanisms sharing a common cause, such as poor dietand/or sedentary lifestyle. Health complications fall into two majorcategories, including those caused by the increased fat mass or anincreased number of fat cells. Osteoarthritis and obstructive sleepapnea are examples of complications due to increased fat mass. Diabetes,cancer, cardiovascular disease, and non-alcoholic fatty liver diseaseare examples of complications due to increased number of fat cells.

Despite public health efforts to understand and correct environmentalfactors contributing to obesity, extensive research into understandingthe factors contributing to the disease, and significant efforts todevelop pharmaceutical and surgical treatments, obesity remains asignificant public health and policy issue. Indeed, World HealthOrganization predicts that obesity may soon replace more traditionalpublic health concerns such as under nutrition and infectious disease asthe most significant cause of poor health. Given this, there exists along-felt and unmet need to provide treatments for obesity and relateddisorders.

SUMMARY

Provided herein are compounds, compositions, pharmaceuticalformulations, methods of treating and methods of using aPKC inhibitorsfor treating and/or preventing of aPKC abnormalities. In one aspect,described herein is a method of treating or preventing an aPKCabnormality in a subject in need thereof, the method containing thesteps of administering an effective amount of an aPKC inhibitor or aderivative thereof to the subject, where the aPKC inhibitor has aformula according to Formula I:

where each where each R₁, when taken separately, is independentlyselected from the group consisting of: hydrogen, halo, C—C6 alkyl, C2-C6alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl,C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkylsulfanyl, aryl, heteroaryl,aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy,heteroaralkoxy, nitro, cyano, amino, C1-C6 alkylamino, di-(C1-C6alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, (C1-C6 alkoxy)carbonyl,(C1-C6 alkyl)aminocarbonyl, di-(C1-C6 alkyl)aminocarbonyl, arylcarbonyl,aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, and arylsulfonyl, or, when takentogether with the atoms to which they are attached, form aC5-C10-membered aromatic, heterocyclic, fused aromatic, fusedheterocyclic, biaromatic, or bihetereocyclic ring, and

where R₂ is selected from the group consisting of: hydrogen, halo, C1-C6alkyl, tert-butyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy,C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkyl furan, C2-C6 alkenyl furan,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, arylsulfonyl, and 2-acetyl-3-oxobutanimidoyl cyanide.

In some embodiments of the method of treating or preventing an aPKCabnormality in a subject in need thereof described above, the derivativeof Formula I is a salt or alcohol of Formula I. In further embodimentsof these methods, the R₂ of Formula I is 2-propylfuran,(E)-2-(prop-1-en-1-yl)furan, or tert-butyl. In other embodiments of anyof the described methods of treating or preventing an aPKC abnormalityin a subject in need thereof described above, the R₁ of Formula I,together with the atoms to which they are attached, form benzene. In anyof these methods, the effective amount of Formula I or derivativethereof ranges from about 0.001 mg to about 1,000 mg. The effectiveamount can be administered to the subject in a dosage form formulatedfor oral, vaginal, intravenous, transdermal, subcutaneous,intraperitoneal, or intramuscular administration. In any of theseembodiments, the aPKC abnormality treated by administration of acompound according to Formula I or a derivative thereof to a subject inneed thereof is obesity, glucose intolerance, metabolic syndrome,hyperinsulinemia, hepatosteatosis, non-alcoholic cirrhosis,hypertriglyceridemia, hypercholesterolemia, polycystic ovary disease,and Alzheimer's disease.

In another aspect, provided is a method for treating or preventing anaPKC abnormality containing the steps of contacting a hepatic cell withan effective amount of an aPKC inhibitor or a derivative thereof, wherethe aPKC inhibitor has a formula according to Formula I:

where each R₁, when taken separately, is independently selected from thegroup consisting of: hydrogen, halo, C—C6 alkyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl, or, when taken together with the atomsto which they are attached, form a C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring, and

where R₂ is selected from the group consisting of: hydrogen, halo, C1-C6alkyl, tert-butyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy,C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkyl furan, C2-C6 alkenyl furan,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, arylsulfonyl, and 2-acetyl-3-oxobutanimidoyl cyanide.

In some embodiments of the method of treating or preventing an aPKCabnormality, described above, the derivative of Formula I is a salt oralcohol of Formula I. In further embodiments of these methods, the R₂ ofFormula I is 2-propylfuran, (E)-2-(prop-1-en-1-yl)furan, or tert-butyl.In other embodiments of any of the described methods of treating orpreventing an aPKC abnormality described above, the R₁ of Formula I,together with the atoms to which they are attached, form benzene. In anyof these methods, the effective amount of Formula I or derivativethereof ranges from about 0.001 mg to about 1,000 mg. The effectiveamount can be administered to the subject in a dosage form formulatedfor oral, vaginal, intravenous, transdermal, subcutaneous,intraperitoneal, or intramuscular administration. In any of theseembodiments, the aPKC abnormality treated by contacting a hepatic cellwith an effective amount of an aPKC inhibitor, where the aPKC inhibitorhas a Formula according to Formula I or a derivative thereof is obesity,glucose intolerance, metabolic syndrome, hyperinsulinemia,hepatosteatosis, non-alcoholic cirrhosis, hypertriglyceridemia,hypercholesterolemia, polycystic ovary disease, and Alzheimer's disease.

In a further aspect, provide is a kit containing an effective amount ofaPKC inhibitor or a derivative thereof provided in a dosage form, wherethe aPKC inhibitor has a Formula according to Formula 1:

where each R₁, when taken separately, is independently selected from thegroup consisting of: hydrogen, halo, C—C6 alkyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl, or, when taken together with the atomsto which they are attached, form a C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring, and

where R₂ is selected from the group consisting of: hydrogen, halo, C1-C6alkyl, tert-butyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy,C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkyl furan, C2-C6 alkenyl furan,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, arylsulfonyl, and 2-acetyl-3-oxobutanimidoyl cyanide;and

instructions fixed in a tangible medium of expression, where theinstructions provide directions for administering the aPKC inhibitor ora derivative thereof to a subject having an aPKC abnormality.

In some embodiments of this aspect, the dosage form is formulated fororal, vaginal, intravenous, transdermal, subcutaneous, intraperitoneal,or intramuscular administration. In additional embodiments of thisaspect, the effective amount of the aPKC inhibitor ranges 15 from about0.001 mg to about 1,000 mg. In other embodiments of the kit according tothis aspect, the aPKC abnormality is obesity, glucose intolerance,metabolic syndrome, hyperinsulinemia, hepatosteatosis, non-alcoholiccirrhosis, hypertriglyceridemia, hypercholesterolemia, polycystic ovarydisease, or Alzheimer's disease.

In another aspect, provided herein are pharmaceutical formulations fortreating or preventing an aPKC abnormality in a subject in need thereofcontaining an effective amount of aPKC inhibitor or a derivative; and

a pharmaceutically acceptable carrier, where the aPKC inhibitor has aformula according to Formula I:

where each R₁, when taken separately, is independently selected from thegroup consisting of: hydrogen, halo, C—C6 alkyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl, or, when taken together with the atomsto which they are attached, form a C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring, and

where R₂ is selected from the group consisting of: hydrogen, halo, C1-C6alkyl, tert-butyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy,C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkyl furan, C2-C6 alkenyl furan,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, arylsulfonyl, and 2-acetyl-3-oxobutanimidoyl cyanide.

In some embodiments of this aspect, the derivative of Formula I is asalt or an alcohol of Formula I. In further embodiments of this aspect,R₂ of Formula I is 2-propylfuran, (E)-2-(prop-1-en-1-yl)furan ortert-butyl. In other embodiments of this aspect, the R₁ of Formula Itogether with the atoms to which they are attached form benzene. Inadditional embodiments of this aspect, the effective amount ranges fromabout 0.001 mg to about 1,000 mg. In further embodiments of this aspect,the aPKC abnormality is selected from the group consisting of obesity,glucose intolerance, metabolic syndrome, hyperinsulinemia,hepatosteatosis, non-alcoholic cirrhosis, hypertriglyceridemia,hypercholesterolemia, polycystic ovary disease, and Alzheimer's disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIG. 1 shows dose-related effects of ACPD on PIP3-stimulated activitiesof PKC-ι (FIG. 1A) and PKC-ζ (FIG. 1B). Recombinant preparations ofPKC-ι and PKC-ζ were 35 incubated with 10 fmol/l PIP3, indicatedconcentrations of ACPD, and other components of the aPKC assay systemand examined for ³²PO₄-labeling of substrate. Values are mean±SEM of 4determinations.

FIG. 2 shows dose-related effects of ACPD on Phorbol Myristoyl Acetate(PMA)-±CaCl₂-stimulated Activities of PKC-α (FIG. 2A), PKC-β2 (FIG. 2B),PKC-δ (FIG. 2C) and PKC-ε (FIG. 2D). Recombinant preparations ofindicated PKCs were incubated with 100 nmol/l PMA±1 mmol/l CaCl₂, asindicated, and indicated concentrations of ACPD and other components ofthe PKC assay system, and examined for ³²PO₄-labeling of substrate.Values are Mean±SEM of 4 determinations.

FIG. 3 shows dose-related effects of ACPD on Activities of Total aPKC(FIG. 3A) and Akt2 (FIG. 3B) in control and insulin-stimulatedhepatocytes of non-diabetic humans. Hepatocytes were incubated for 24hours±1 μmol/l insulin and indicated concentrations of ACPD and examinedfor immunoprecipitable activities of total aPKC and Akt2. Values aremean±SEM of 4 determinations.

FIG. 4 shows the effect of ACPD on expression of lipogenic andgluconeogenic factors in basal and insulin-stimulated hepatocytes ofnon-diabetic and type 2 diabetic (T2DM) humans. mRNA levels of SREBP-1c(FIG. 4A), PEPCK (FIG. 4B), FAS (FIG. 4C), and G6Pase (FIG. 4D) weremeasured in hepatocytes of non-diabetic and T2DM humans treated for 24hours without (0) or with indicated concentrations of ACPD, in thepresence (solid bars) and absence (open bars) of about 1 μmol/l insulin.Relative values are mean±SEM of 5 determinations. Symbols indicate: *,P<0.05, ACPD-treated basal or ACPD-treated+insulin-stimulated valueversus corresponding basal or insulin-stimulated value of theACPD-untreated (0) group; †, P<0.05, insulin-treated value of the 0group versus basal value of the 0 group; and ‡, P<0.05, basal orinsulin-stimulated diabetic value of the 0 group versus basal orinsulin-stimulated non-diabetic value of the 0 group.

FIG. 5 shows the effect of ACPD on resting/basal and insulin-stimulatedaPKC activity in mouse liver (FIG. 5A) and muscle (FIG. 5B). Normal micewere treated for 24, 48, or 72 hours with a single subcutaneousinjection of ACPD (10 mg/kg body weight), and, at 24, 48, or 72 hours,the mice were injected intraperitoneally with 1 U/kg body weight insulinand killed 15 minutes later. Tissues were examined for immunprecipitabletotal aPKC activity. Values are mean±SEM of 4 determinations.

FIG. 6 shows the effect of high fat feeding (HFF) and ICAP or ACPD (s.c.injection of ICAP, 1 mg/kg body weight, or ACPD; 10 mg/kg body weight)on basal and insulin-stimulated activity of aPKC (FIGS. 6A and 6B) andAkt2 (FIGS. 6C and 6D) in muscle (FIGS. 6B and 6D) and liver (FIGS. 6Aand 6C). After 10 weeks of feeding a low fat diet (10% calories fromfat) or a high fat diet (40% of calories from milk fat) and treatmentwith or without ICAP or ACPD, as indicated, mice were treated for 15minutes with insulin (1 U/kg body weight given intraperitoneally) andthen killed. Liver and muscle tissues were analyzed forimmunoprecipitable aPKC or Akt activity. Values are mean±SEM of 6determinations. Asterisks indicate: *, P<0.05: **; P<0.01; and ***,P<0.001.

FIG. 7 demonstrates the effects of HFF diet and ICAP (I) or ACPD (A) onhepatic resting/basal and insulin stimulated phosphorylation ofSer⁴⁷³-Akt (FIG. 7A), Ser9-GSK3β (FIG. 7B), Thr^(555/560)-PKC-λ/ζ, (FIG.7C), and Thr²⁴⁴⁸-mTOR (FIG. 7D). Over a period of 10 weeks, mice werefed a low-fat (10% of calories from fat) (L) or high-fat (40% ofcalories from milk fat) (H) diets and treated with or without ICAP orACPD and fed mice were treated for 15 min before killing with or withoutinsulin (1 unit/kg body weight i.p.). The liver was harvested andexamined for immunoreactivity of indicated signaling factors. Bar valuesare mean±SEM (n=6). *P<0.05; **P<0.01; ***P<0.001 for indicatedcomparisons. Letters above bars indicated the following: a, P<0.05; b,P<0.01; c, P<0.001 for insulin-stimulated versus resting/basal values incorresponding treatment groups. Representative immunoblots are shown forindicated phosphor-proteins and GAPDH loading controls

FIG. 8 demonstrates the effects of a HFF diet and ICAP (I) or ACPD (A)on mRNA 15 levels of lipogenic enzymes SREBP-1c (FIG. 8A) and FAS (FIG.8B), as well as mRNA levels of gluconeogenic enzymes PEPCK (FIG. 8C) andG6Pase (FIG. 8D) in the livers of ad libitum-fed mice. Over a period of10 weeks, mice consumed low-fat (L) and high-fat (H) diets and treatedwith or without ICAP or ACPD. After killing, liver tissue was examinedfor mRNA of the indicated enzymes. Values are means±SEM (n=12). Acute 15minute 20 insulin treatment prior to killing, as described with respectto FIG. 7, did not alter mRNA levels and data is not shown for thistreatment group. *P<0.05; **P<0.01; ***P<0.001 for indicatedcomparisons.

FIG. 9 demonstrates the effects of a HFF diet and ICAP (I) or ACPD (A)on immunoreactive protein levels of lipogenic enzymes SREBP-1c (FIG. 9A)and FAS (FIG. 9B), as well as immunoreactive protein levels ofgluconeogenic enzymes PEPCK (FIG. 9C) and G6Pase (FIG. 9D) in the liversof ad libitum-fed mice. Over a 10 week period, mice consumed either alow-fat (L) or a high-fat (H) diet and were treated with or without ICAPor ACPD. After killing, liver tissue was examined for immunoreactiveprotein of the indicated enzymes. Values are means±SEM (n=12). An acute15-minute insulin treatment, as described with respect to FIG. 7, didnot alter immunoreactive protein levels. This data is not shown.*P<0.05; **P<0.01; ***P<0.001 for indicated comparisons. Representativeimmunoblots are shown for indicated proteins. The levels of the activeSREBP-1c fragment were measured in nuclear preparations.

FIG. 10 demonstrates the effects of a HFF diet and ICAP (I) or ACPD (A)on phosphorylation of Ser256-FoxO1 in liver lysates (FIG. 10A); recoveryof immunoreactivity of aPKC, FoxO1, and WD40/ProF in WD40/ProFimmunoprecipitates (FIG. 10B); recovery of Akt enzyme activity inWD40/ProF immunoprecipitates (FIG. 10C); and recovery of aPKC enzymeactivity in WD40/ProF immunoprecipitates (FIG. 10D). Over a time periodof 10 weeks, mice were fed a low-fat (L) or high-fat (H) diet andtreated with or without ICAP or ACPD. 15 minutes before killing, micewere treated with or without insulin (1 unit/kg body wt, injectedintraperitoneally). The liver was harvested and analyzed for indicatedsignaling factors in liver lysates or WD40/ProF immunoprecipitatesprepared from liver lysates. Bargram values are mean±SEM of sixdeterminations. *P<0.05; **P<0.01; ***P<0.001 for indicated comparisons.Letters above bars indicate the following: a, P<0.05; b, P<0.01; and c,P<0.001 for insulin-stimulated versus basal/resting values incorresponding treatment groups. FoxO1 and WD40/ProF levels were notaltered by treatments. Large amounts of immunoreactive immuno-gglobulins precluded accurate measurement of nearby Akt in WD40/ProFimmunoprecipitates.

FIG. 11 shows the effects of high fat feeding and ICAP or ACPD onglucose tolerance (FIG. 11A) and serum levels of glucose in fed mice(FIG. 11B), serum insulin in fasted mice (FIG. 11C), serum levels oftriglycerides in fed mice (FIG. 11D), and serum levels of cholesterol infed mice (FIG. 11E). Mice were fed a low fat diet (10% calories fromfat) or a high fat diet (40% of calories from milk fat) andsimultaneously treated with or without ICAP or ACPD. At 9 weeks oftreatment, mice were subjected to an overnight fast followed by glucosetolerance testing (2 mg glucose/kg body weight injectedintraperitoneally) with blood glucose levels measured at 0, 30, 60, 90and 120 minutes. After 10 weeks, mice were injected with or withoutinsulin (1 U/kg body weight given intraperotoneally) and were thenkilled 15 minutes post injection. Serum was analyzed for insulin,triglycerides and cholesterol. Values are mean±SEM of 12 determinationsfor glucose tolerance testing, and mean±SEM of 6 determinations foreffects of insulin on serum glucose. Asterisks indicate: *, P<0.05: **;P<0.01; and ***, P<0.001. Data regarding treatment with insulin is notshown where no significant differences between insulin treated andnon-insulin treated mice were observed.

FIG. 12 shows the effects of high fat feeding and ICAP or ACPD on bodyweight (FIG. 12A), change in body weight (FIG. 12B) levels of hepatictriglycerides (FIG. 12C), weights of epididymal plus retroperitoneal fatpads (FIG. 12D), and weekly food intake (FIG. 12E). Mice were fed a lowfat diet (10% calories from fat) or a high fat diet (40% of caloriesfrom milk fat) and treated with or without ICAP or ACPD. Body weight andfood intake was measured weekly. After 10 weeks, the mice were killed,and tissues were analyzed for indicated parameters. Values are mean±SEMof 12 determinations. Asterisks indicate: *, P<0.05: **; P<0.01; and***, P<0.001.

FIG. 13 demonstrates the effects of an HFF diet and ICAP (I) or ACPD (A)on hepatic fat contents as per Oil Red O staining in mice consuming alow-fat diet (FIG. 13A), a high fat diet (FIG. 13B), a high-fat diet andtreated with ICAP (FIG. 13C), and a high fat-diet and treated with ACPD(FIG. 13D). Over a period of 10 weeks, mice were consuming low-fat andhigh-fat diets and treated with or without ICAP or ACPD.

FIG. 14 demonstrates the effects of ceramide on aPKC immunoprecipitatedfrom liver lysates obtained from mice consuming low-fat (LF) andhigh-fat (HF) diets for 10 weeks and treated with or without insulin(Ins) for 15 min prior to killing, as described in FIG. 7 (except thatthese mice were not treated with an aPKC inhibitor). Activity of aPKCwith indicated concentrations of ceramide (Sigma) was measured. Valuesare mean±SEM of three to five determinations.

FIG. 15 demonstrates the effects of the feeding of HFF and LFF onhepatic levels of 10 ceramide and sphingomyelin species. Over a periodof 10 weeks, mice were fed a low-fat or high-fat diet but were nottreated with aPKC inhibitors. Bargram values are mean±SEM of eightdeterminations. DH, dihydro; SM, sphingomyelin.

FIG. 16 shows basal and insulin-stimulated phosphorylation of aPKC andAkt in liver (FIGS. 16A and 16C) and muscle (FIGS. 16B and 16D) of leancontrol (ob⁺) mice and ob/ob mice treated without or with ACPD. Leancontrol (ob⁺) mice and ob/ob mice were injected subcutaneously oncedaily for 10 weeks with saline vehicle or ACPD (10 mg/kg body weightgiven subcutaneously) in saline vehicle, as indicated, and treatedacutely with (+) or without (−) insulin (1 U/kg body weight) for 15 minprior to killing. Tissues were subjected to Western analyses forp-thr555/560-PKC-ι/ζ/λ and p-ser-473-Akt to assess respective kinaseactivities. Representative blots are shown. Blots are representative of5-6 determinations.

FIG. 17 shows the effects of ACPD on glucose tolerance in ob/ob mice.Lean control (ob⁺) mice and ob/ob mice were injected subcutaneously oncedaily for 10 weeks with saline vehicle or ACPD (10 mg/kg body weightgiven subcutaneously) in saline vehicle, as indicated, and, during the9^(th) week, after an overnight fast, the mice were subjected to glucosetolerance testing (2 mg glucose/kg body weight administeredintraperitoneally). At the indicated times, tail vein blood was obtainedfor determination of serum levels of glucose (main panel) and insulin(inset). Values are mean±SEM of 10-12 determinations. Asterisksindicate: *, P<0.05; **, P<0.01; and ***, P<0.001.

FIG. 18 demonstrates the effect of ACPD on body weight (FIG. 18A), foodintake 30 (FIG. 18B), combined weight of epididymal plus retroperitonealfat depots (FIG. 18C), serum triglycerides (FIG. 18D), and livertriglycerides (FIG. 18E) in ob/ob mice. Mice were treated with ACPDdaily for 10 weeks. Insulin treatment 15 minutes prior to killing didnot alter triglyceride levels. Values are mean±SEM of (N)determinations. Asterisks: *, P<0.05; ** P<0.01; ***, P<0.001.

FIG. 19 demonstrates the effects of ACPD on phosphorylation/activitiesof pSer⁴⁷³-Akt (FIGS. 19A and 19B) and p-Thr^(556/560)-PKC-λ/ζ, (FIGS.19C and 19D) in resting/basal and insulin-stimulated conditions andduring treatment with an ACPD in liver (FIGS. 19A and 19C) and muscle(FIGS. 19B and 19D) lysates of lean ob⁺ and obese-phase ob/ob mice. Over10 weeks, lean ob⁺ mice and ob/ob mice were injected subcutaneouslydaily with 0.2 ml physiologic saline or saline containing ACPD (10 mg/kgbody weight). Before killing, ad libitum fed mice were treated for 15min±insulin (1 U/kg body-weight, intraperitoneally). Values are mean±SEMof (N) determinations. Asterisks: *, P<0.05: **, P<0.01; ***, P<0.001.

FIG. 20 demonstrates phosphorylation of Akt substrates, FoxO1 (FIG.20A), mTOR (FIG. 20B) and GSK3β (FIG. 20C). Association of aPKC (FIG.20D) and Akt (FIG. 20E) with hepatic WD40/ProF in livers of lean ob⁺ andobese-phase ob/ob mice during 10 resting/basal and insulin-stimulatedconditions and during treatment with ACPD is also demonstrated in FIG.20. Mice were treated with ACPD and insulin as in FIG. 19. In FIGS. 20A,20B and 20C, liver lysates were blotted directly. In FIGS. 20D and 20E,WD40/ProF was immunoprecipitated from liver lysates, and portions ofimmunoprecipitates were blotted. Values are mean±SEM of (N)determinations. Asterisks: *, P<0.05: **, P<0.01; ***, P<0.001.Representative blots are shown; note unchanged level s of FoxO1 inlysates and WD40/ProF in immunoprecipitates.

FIG. 21 demonstrates the effects of treatment with aPKC inhibitor, ACPD,on mRNA levels of hepatic SREBP-1c (FIG. 21A), FAS (FIG. 21B), ACC (FIG.21C), TNF-α (FIG. 21D), PEPCK (FIG. 21E) and G6Pase (FIG. 21F) in obeseob/ob mice. Mice were treated with ACPD and insulin as 19. 15-mininsulin treatment did not alter mRNA or protein levels of these enzymes.Values are mean±SEM of (N) determinations. Asterisks: *, P<0.05: **,P<0.01; ***, P<0.001.

FIG. 22 demonstrates the effects of treatment with aPKC inhibitor, ACPD,on hepatic nuclear levels of the active 60-70 kDa proteolytic fragmentof SREBP-1c (FIG. 22A) and 25 the active p65/RelA subunit of NFκB (FIG.34B) in obese ob/ob mice. Mice were treated with ACPD and insulin priorto killing as previously described 15-min insulin treatment did notalter mRNA or protein levels of these enzymes. Values are mean±SEM of(N) determinations. Asterisks: *, P<0.05: **, P<0.01; ***, P<0.001.Representative blots are shown.

FIG. 23 demonstrates the effects of treatment with aPKC inhibitor, ACPD,on hepatic lysate protein levels of ACC (FIG. 23A), FAS (FIG. 23B),PEPCK (FIG. 23C), and G6Pase (FIG. 23D) in obese ob/ob mice. Mice weretreated with ACPD and insulin as in FIGS. 22. 15-min insulin treatmentdid not alter mRNA or protein levels of these enzymes. Values aremean±SEM of (N) determinations. Asterisks: *, P<0.05: **, P<0.01; ***,P<0.001. Representative blots are shown.

FIG. 24 is a schematic showing factors involved in early development ofhepatic insulin resistance in diet-induced obesity.

FIG. 25 demonstrates the resting/basal and insulin-stimulated activitiesof aPKC (FIGS. 25A and 25C) and Akt2 (FIGS. 25B and 25D) in the brainsof control (Con) and high-fat fed (HFF) mice (FIGS. 25A and 25B) and inthe brains of control (Con) and ob/ob (OB) mice (FIGS. 25C and 25D). HFFmice were fed a diet containing 40% of calories derived from milk fatsand all other mice were fed standard mice chow containing 10% fat. Allmice were fed these diets over a 10 week period. Where indicated (+),mice were acutely treated with insulin (1 U/kg body weight) that wasadministered intraperitoneally 15 minutes prior to killing.Activity/activation was assessed by Western blot analyses ofphospho-protein immunoreactivity and quantitative scanning of the blots.The results were normalized to an endogenous control. Values of scansare shown as the mean±SEM of 4-6 determinations. Asterisks indicatedP<0.05 (as per ANOVA) versus the basal control.

FIG. 26 demonstrates the resting/basal and insulin-stimulatedphosphorylation of Akt substrates FoxO1 (FIGS. 26A and 26C) and FoxO3a(FIGS. 26B and 26D) in the brains of control (Con) and high-fat fed(HFF) mice (FIGS. 26A and 26B) and in the brains of 15 control (Con) andob/ob (OB) mice (FIGS. 26C and 26D). HFF mice were fed a diet containing40% of calories derived from milk fats and all other mice were fedstandard mice chow containing 10% fat. All mice were fed these dietsover a 10 week period. Where indicated (+), mice were acutely treatedwith insulin (1 U/kg body weight) that was administeredintraperitoneally 15 minutes prior to killing. Levels ofphospho-proteins were assessed by Western blot analyses usingphospho-peptide-specific antisera followed by quantitative scanning ofthe blots. The results were normalized to an endogenous control.

Values of scans are shown as the mean±SEM of 4-6 determinations.Asterisks indicated P<0.05 (as per ANOVA) versus the basal control.

FIG. 27 demonstrates the resting/basal and insulin-stimulatedphosphorylation of Akt substrates GSK3β (FIGS. 27A and 27C) and mTOR(FIGS. 27B and 27D) in the brains of control (Con) and high-fat fed(HFF) mice (FIGS. 27A and 27B) and in the brains of control (Con) andob/ob (OB) mice (FIGS. 27C and 27D). HFF mice were fed a diet containing40% of calories derived from milk fats and all other mice were fedstandard mice chow containing 10% fat. All mice were fed these dietsover a 10 week period. Where indicated 30 (+), mice were acutely treatedwith insulin (1 U/kg body weight) that was administeredintraperitoneally 15 minutes prior to killing. Levels ofphospho-proteins were assessed by Western blot analyses usingphospho-peptide-specific antisera followed by quantitative scanning ofthe blots. The results were normalized to an endogenous control. Valuesof scans are shown as the mean±SEM of 4-6 determinations. Asterisksindicated P<0.05 (as per ANOVA) versus the basal control.

FIG. 28 demonstrates the resting/basal and insulin-stimulated activitiesof aPKC (in the brains of control (Con) versus heterozygousmuscle-specific PKC-λ knockout (KO) mice. All mice were fed standardchow containing 10% fat. Where indicated (+), mice were acutely treatedwith insulin (1 U/kg body weight) that was administeredintraperitoneally 15 minutes prior to killing. Where indicated, micewere treated for 8 days with ICAPP. Activity/activation of aPKC wasassessed by Western blot analyses of phospho-protein immunoreactivityfollowed by quantitative scanning of the blots. The results werenormalized to an endogenous control. Values of scans are the mean±SEM of4-6 determinations. Asterisks indicate P<0.05 (as per ANOVA) versus thebasal control.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to 15 the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated 20 range. Where thestated range includes one or both of the limits, ranges excluding eitheror both of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, organic chemistry, biochemistry, botany and the like,which are within the skill of the art. Such techniques are explainedfully in the literature.

DEFINITIONS

As used herein, “derivative” refers to any compound having the same or asimilar core structure to the compound but having at least onestructural difference, including substituting, deleting, and/or addingone or more atoms or functional groups. The term “derivative” does notmean that the derivative is synthesized from the parent compound eitheras a starting material or intermediate, although this may be the case.The term “derivative” can include salts, prodrugs, or metabolites of theparent compound. Derivatives include compounds in which free aminogroups in the parent compound have been derivatized to form aminehydrochlorides, p-toluene sulfoamides, benzoxycarboamides,t-butyloxycarboamides, thiourethane-type derivatives,trifluoroacetylamides, chloroacetylamides, or formamides. Derivativesinclude compounds in which carboxyl groups in the parent compound havebeen derivatized to form salts, methyl and ethyl esters, or other typesof esters or hydrazides. Derivatives include compounds in which hydroxylgroups in the parent compound have been derivatized to form O-acyl orO-alkyl derivatives. Derivatives include compounds in which a hydrogenbond donating group in the parent compound is replaced with anotherhydrogen bond donating group such as OH, NH, or SH. Derivatives includereplacing a hydrogen bond acceptor group in the parent compound withanother hydrogen bond acceptor group such as esters, ethers, ketones,carbonates, tertiary amines, imine, thiones, sulfones, tertiary amides,and sulfides. “Derivatives” also includes extensions of the replacementof the cyclopentane ring with saturated or unsaturated cyclohexane orother more complex, e.g., nitrogen-containing rings, and extensions ofthese rings with side various groups

As used herein, “active derivative” and the like means a derivativecompound that retains an ability to inhibit or reduce the activity oneor more PKCs, PKC-zeta, and PKC-35 lambda/iota of a subject to which itis administered, as compared uninhibited (or normal PKCs).

As used herein, “administering” refers to an administration that isoral, topical, intravenous, subcutaneous, transcutaneous, transdermal,intramuscular, intra-joint, parenteral, intra-arteriole, intradermal,intraventricular, intracranial, intraperitoneal, intralesional,intranasal, rectal, vaginal, by inhalation, or via an implantedreservoir. The term “parenteral” includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, and intracranial injections orinfusion techniques.

As used herein, “composition” refers to a combination of an activeagent(s) and another compound or composition, inert (for example, adetectable agent or label) or active, such as an adjuvant.

As used herein, “control” is an alternative subject or sample used in anexperiment for comparison purposes and included to minimize ordistinguish the effect of variables other than an independent variable.A control can be positive or negative.

As used herein, “concentrated” refers to an amount of a molecule,compound, or composition, including, but not limited to, a chemicalcompound, polynucleotide, peptide, polypeptide, protein, antibody, orfragments thereof, that indicates that the sample is distinguishablefrom its naturally occurring counterpart in that the concentration ornumber of molecules per volume is greater than that of its naturallyoccurring counterpart.

As used herein, “diluted” refers to an amount of a molecule, compound,or composition including but not limited to, a chemical compound,polynucleotide, peptide, polypeptide, protein, antibody, or fragmentsthereof, that indicates that the sample is distinguishable from itsnaturally occurring counterpart in that the concentration or number ofmolecules per volume is less than that of its naturally occurringcounterpart.

As used herein, “pharmaceutical formulation” refers to the combinationof an active agent, compound, or ingredient with a pharmaceuticallyacceptable carrier or excipient, making the composition suitable fordiagnostic, therapeutic, or preventive use in vitro, in vivo, or exvivo.

As used interchangeably herein, “subject,” “individual,” or “patient,”refers to a vertebrate, preferably a mammal, more preferably a human.Mammals include, but are not limited to, murines, simians, humans, farmanimals, sport animals, and pets. The term “pet” refers to a dog, cat,guinea pig, mouse, rat, rabbit, ferret, and the like. The term “farmanimal” refers to a horse, sheep, goat, chicken, pig, cow, donkey,llama, alpaca, turkey, and the like.

As used interchangeably herein, “biocompatible” and “biologicallycompatible” refer to materials that are, with any metabolites ordegradation products thereof, generally non-toxic to the recipient, andcause no significant adverse effects to the recipient. Generallyspeaking, biocompatible materials are materials which do not elicit asignificant inflammatory or immune response when administered to apatient. In some embodiments, a biocompatible material elicits nodetectable change in one or more biomarkers indicative of an immuneresponse. In some embodiments, a biocompatible material elicits nogreater than a 10% change, no greater than a 20% change, or no greaterthan a 40% change in one or more biomarkers indicative of an immuneresponse.

As used herein, “therapeutic” refers to curing or treating a symptom ofa disease or condition.

As used herein, “preventative,” “preventing,” “prevent” and the likerefer to partially or completely delaying or precluding the onset orrecurrence of a disorder or conditions and/or one or more of itsattendant symptoms or barring a subject from acquiring or reacquiring adisorder or condition or reducing a subject's risk of acquiring orreacquiring a disorder or condition or one or more of its attendantsymptoms.

As used herein, “mitigate” refers to reducing a particularcharacteristic, symptom, or other biological or physiological parameterassociated with a disease or disorder.

As used herein, “separated” refers to the state of being physicallydivided from the original source or population such that the separatedcompound, agent, particle, chemical compound, or molecule can no longerbe considered part of the original source or population.

As used herein, “tangible medium of expression” refers to a medium thatis physically tangible and is not a mere abstract thought or anunrecorded spoken word. Tangible medium of expression includes, but isnot limited to, words on a cellulosic or plastic material or electronicdata stored on a suitable device such as a flash memory or CD-ROM.

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within +−10% of the indicated value, whichever is greater.

As used herein, “synergistic effect,” “synergism,” or “synergy” refersto an effect arising between two or more molecules, compounds,substances, factors, or compositions that that is greater than ordifferent from the sum of their individual effects.

As used herein, “additive effect” refers to an effect arising betweentwo or more molecules, compounds, substances, factors, or compositionsthat is equal to or the same as the sum of their individual effects.

As used herein, “mammal” refers to any animal classified as a mammal,including a human, domestic and farm animals, nonhuman primates, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

As used herein, “pharmaceutically acceptable” refers to compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio, in accordance with the guidelines ofagencies such as the Food and Drug Administration.

The terms “sufficient” and “effective,” as used interchangeably herein,refer to an amount (e.g. mass, volume, dosage, concentration, and/ortime period) needed to achieve one or more desired result(s). Forexample, a therapeutically effective amount refers to an amount neededto achieve one or more therapeutic effects.

As used herein, “pharmaceutically acceptable carrier or excipient”refers to a carrier or excipient that is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable, and includes a carrier orexcipient that is acceptable for veterinary use as well as humanpharmaceutical use. A “pharmaceutically acceptable carrier or excipient”as used herein also includes both one and more than one such carrier orexcipient. Pharmaceutically acceptable carriers include, but are notlimited to, diluents, preservatives, binders, lubricants,disintegrators, swelling agents, fillers, stabilizers, and combinationsthereof.

As used herein, “pharmaceutically acceptable salts” refers to any acidor base addition salt whose counter-ions are non-toxic to the subject towhich they are administered in pharmaceutical doses of the salts.Specific examples of pharmaceutically acceptable salts are known tothose of ordinary skill in the art.

As used herein, “subject” is defined herein to include animals such asmammals, including, but not limited to, primates (e.g., humans), cows,sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. Inpreferred embodiments, the subject is a human.

As used herein, “suitable substituent” means a chemically andpharmaceutically acceptable group, i.e., a moiety that does notsignificantly interfere with the preparation of or negate the efficacyof the inventive compounds. Such suitable substituents may be routinelychosen by those skilled in the art. Suitable substituents include butare not limited to the following: a halo, C1-C6 alkyl, C2-C6 alkenyl,C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, 35(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl. The groups listed above as suitablesubstituents are as defined hereinafter except that a suitablesubstituent may not be further optionally substituted.

As used herein, “optically substituted” indicates that a group may beunsubstituted or substituted with one or more substituents as definedherein.

The term “alkyl” refers to the radical of saturated aliphatic groups(i.e., an alkane with one hydrogen atom removed), includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,and C₃-C₃₀ for branched chains). In other embodiments, a straight chainor branched chain alkyl contains 20 or fewer, 15 or fewer, or 10 orfewer carbon atoms in its backbone. Likewise, in some embodimentscycloalkyls have 3-10 carbon atoms in their ring structure. In some ofthese embodiments, the cycloalkyl have 5, 6, or 7 carbons in the ringstructure.

The term “alkyl” (or “lower alkyl”) as used herein is intended toinclude both “unsubstituted alkyls” and “substituted alkyls,” the latterof which refers to alkyl moieties having one or more substituentsreplacing a hydrogen on one or more carbons of the hydrocarbon backbone.Such substituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oran aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons in its backbone structure. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl,” as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl, and—S-alkynyl. Representative alkylthio groups include methylthio,ethylthio, and the like. The term “alkylthio” also encompassescycloalkyl groups, alkene and cycloalkene groups, and alkyne groups.“Arylthio” refers to aryl or heteroaryl groups. Alkylthio groups can besubstituted as defined above for alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy,” as used herein, refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl is an ether or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O— alkenyl, and —O-alkynyl. The terms“aroxy” and “aryloxy”, as used interchangeably herein, can berepresented by —O-aryl or O-heteroaryl, wherein aryl and heteroaryl areas defined below. The alkoxy and aroxy groups can be substituted asdescribed above for alkyl.

The terms “amine” and “amino” (and its protonated form) areart-recognized and refer to both unsubstituted and substituted amines,e.g., a moiety that can be represented by the general formula:

wherein R, R′, and R″ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH2)_(m)-R_(C) or R and R′ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R_(C) represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R or R′ can bea carbonyl, e.g., R, R′ and the nitrogen together do not form an imide.In other embodiments, the term “amine” does not encompass amides, e.g.,wherein one of R and R′ represents a carbonyl. In further embodiments, Rand R′ (and optionally R″) each independently represent a hydrogen, analkyl or cycloakly, an alkenyl or cycloalkenyl, or alkynyl. Thus, theterm “alkylamine” as used herein means an amine group, as defined above,having a substituted (as described above for alkyl) or unsubstitutedalkyl attached thereto, i.e., at least one of R and R′ is an alkylgroup.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R and R′ are as defined above.

As used herein, “Aryl” refers to C₅-C₁₀-membered aromatic, heterocyclic,fused aromatic, fused heterocyclic, biaromatic, or bihetereocyclic ringsystems. Broadly defined, “aryl”, as used herein, includes 5-, 6-, 7-,8-, 9-, and 10-membered single-ring aromatic groups that may includefrom zero to four heteroatoms, for example, benzene, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like. Those aryl groups havingheteroatoms in the ring structure may also be referred to as “arylheterocycles” or “heteroaromatics.” The aromatic ring can be substitutedat one or more ring positions with one or more substituents including,but not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, alkoxyl, amino (or quaternized amino), nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, andcombinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl.”

The term “aralkyl,” as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aralkyloxy” can be represented by —O-aralkyl, wherein aralkylis as defined above.

The term “carbocycle,” as used herein, refers to an aromatic ornon-aromatic ring(s) in which each atom of the ring(s) is carbon.

“Heterocycle” or “heterocyclic,” as used herein, refers to a monocyclicor bicyclic structure containing 3-10 ring atoms, and in someembodiments, containing from 5-6 ring atoms, wherein the ring atoms arecarbon and one to four heteroatoms each selected from the followinggroup of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or isH, O, (C₁-C₁₀) alkyl, phenyl or benzyl, and optionally containing 1-3double bonds and optionally substituted with one or more substituents.Examples of heterocyclic rings include, but are not limited to,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.Heterocyclic groups can optionally be substituted with one or moresubstituents at one or more positions as defined above for alkyl andaryl, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R and R′are as defined above. Where X is an oxygen and R or R′ is not hydrogen,the formula represents an “ester”. Where X is an oxygen and R is asdefined above, the moiety is referred to herein as a carboxyl group, andparticularly when R is a hydrogen, the formula represents a “carboxylicacid.” Where X is an oxygen and R′ is hydrogen, the formula represents a“formate.” In general, where the oxygen atom of the above formula isreplaced by sulfur, the formula represents a “thiocarbonyl” group. WhereX is a sulfur and R or R′ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R is not hydrogen, the above formula represents a“ketone” group. Where X is a bond, and R is hydrogen, the above formularepresents an “aldehyde” group.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Exemplary heteroatoms include, but are notlimited to, boron, nitrogen, oxygen, phosphorus, sulfur, silicon,arsenic, and selenium.

As used herein, the term “nitro” refers to —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” refers to —SH; theterm “hydroxyl” refers to —OH; and the term “sulfonyl” refers to —SO₂—.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, e.g. 1-14 carbon atoms,and optionally include one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats.

Representative substituents include alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, amino acid, peptide, andpolypeptide groups.

Heteroatoms, such as nitrogen, may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e., a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

As used herein, “effective amount” refers to the amount of an aPKCinhibitor or derivative thereof described herein that will elicit thebiological or medical response of a tissue, system, animal, or humanthat is being sought by the researcher, veterinarian, medical doctor orother clinician. “Effective amount” includes that amount of an aPKCinhibitor or derivative thereof described herein that, whenadministered, is sufficient to prevent development of, alleviate to someextent, one or more of the symptoms of an atypical protein kinase Cenzyme (aPKC) abnormality being treated. The effective amount will varydepending on the exact chemical structure of the aPKC inhibitor orderivative thereof, the severity of the aPKC abnormality, the route ofadministration, the time of administration, the rate of excretion, thedrug combination, the judgment of the treating physician, the dosageform, and the age, weight, general health, sex and/or diet of thesubject to be treated.

The terms “treat,” “treating,” “treatment,” and grammatical variationsthereof as used herein include partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disorder or condition such as an atypicalprotein kinase C enzyme abnormality and/or alleviating, mitigating orimpeding one or more causes of a disorder or condition such as anatypical protein kinase C enzyme abnormality.

Treatments according to the embodiments disclosed herein may be appliedpreventively, prophylactically, pallatively, or remedially. In someinstances, the terms “treat,” “treating,” “treatment,” and grammaticalvariations thereof include partially or completely reducing a conditionor symptom associated with an atypical protein kinase C enzymeabnormality as compared with prior to treatment of the subject or ascompared with the incidence of such condition or symptom in a general orstudy population.

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within +−10% of the indicated value, whichever is greater.

As used herein, “dosage form” or “unit dosage form” refers to apharmaceutical formulation that is administered to a subject in need oftreatment and generally may be in the form of tablets, capsules, sachetscontaining powder or granules, liquid solutions or suspensions, patches,and the like.

As used herein, “aPKC abnormality” refers to any symptom, condition,disease, disorder, syndrome that involves aberrant aPKC activity orfunction. Examples include but are not limited to, obesity, insulinresistance, glucose intolerance, hyperinsulinemia, any form of diabetes,weight gain, metabolic syndrome, Alzheimer's disease, hepatosteatosis,non-alcoholic cirrhosis, hypertriglyceridemia, hypercholesterolemia,polycystic ovary disease, and any disorder, disease, condition, orsymptom arising from local or systemic hyperinsulinemia and/or metabolicsyndrome that arise from abnormal or aberrant aPKC activity or function,including but not limited to, brain disorders such as Alzheimer'sdisease and cardiovascular diseases and disorders.

DISCUSSION

Insulin-resistant states of obesity, metabolic syndrome, and type 2diabetes mellitus (T2DM) are pandemic in Western societies. Insulinresistance implies an impairment in glucose metabolism that initiallyincreases insulin secretion. There is no cure for insulin-resistantstates of obesity, metabolic syndrome, or T2DM. However, diet, exercise,and weight loss may help alleviate symptoms. When these fail, many ofthese individuals rely on insulin therapy or other therapeuticcompounds, many of which have harsh side effects, to regulate bloodglucose levels. As such, there exists a need for improved treatments forinsulin-resistant states of obesity, metabolic syndrome, and T2DM.

Protein kinase C (PKC) enzymes are involved in regulatinggluconeogenesis and lipogenesis and therefore play a role in overallglucose metabolism and insulin sensitivity. In particular, as shown inFIG. 24, aPKC plays a key role in the development of hepatic andsecondary systemic insulin resistance in dietary-induced obesity. Inresponse to dietary excess, availability of lipids that directlyactivate aPKC, e.g., ceramide and phosphatidic acid, increases.Subsequent activation of hepatic aPKC increases binding of aPKC to ProF,a scaffolding protein that couples Akt and FoxO1, and this leads toimpaired ability of Akt2 to phosphorylate FoxO1 on Ser²⁵⁶; as a result,expression of PEPCK and G6Pase and hepatic glucose output increase.Ensuing increases in blood glucose levels stimulate insulin secretion,and both glucose and insulin, as well as fatty acids, increasephosphatidic acid production via the de novo pathway. Increased insulinsecretion activates hepatic Akt2, as well as aPKC, which togetherincrease hepatic lipid production, thereby providing more substrates forphosphatidic acid and ceramide synthesis. In short, a vicious cycle isset up for lipid production and aPKC activation. This cycle is abettedin human (but not rodent) liver by virtue of the fact that increasedaPKC activity provokes increases in levels of PKC-ι mRNA and protein. Asa by-product of increases in circulating levels of liver-derived lipidsand cytokines, insulin signaling in muscle and certain other tissues(e.g., adipose tissue) is impaired, adding further to diminished glucosedisposal and systemic insulin resistance.

As demonstrated herein, atypical PKC (aPKC) inhibitors act to counteraPKC abnormalities, which dysregulate hepatic and secondary insulinsensitivity through altering the pathways involved in gluconeogenesisand lipogenesis. For example, aPKC inhibitors reduce dietary inducedobesity. With that in mind, provided herein are compounds andpharmaceutical formulations for treating an atypical protein kinase Cenzyme abnormality. In some instances the aPKC abnormality results in adisease or condition, such as insulin-resistant obesity, metabolicsyndrome, or diabetes (including T2DM and Type 1).

Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

aPKC Inhibitors and Formulations Thereof

Provided herein are aPKC inhibitors and formulations thereof. An “aPKCinhibitor,” as used herein, refers generally to a molecule that can bindto PKC, and/or PKC A/i and inhibit or reduce the ability of the PKC tofunction relative to a non-inhibited (normal or wild-type) PKC. aPKCinhibitors include derivatives, including but not limited to activederivatives, of the aPKC inhibitors described herein. In someembodiments, the aPKC inhibitor or derivative thereof contains one ormore suitable substituents.

aPKC Inhibitors

The aPKC inhibitor can have a structure according to Formula I or aderivative thereof:

wherein R₁ are each independently selected from the following: hydrogen,halo, C1-C6 alkyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy,C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkylsulfanyl, aryl, heteroaryl,aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy,heteroaralkoxy, nitro, cyano, amino, C1-C6 alkylamino, di-(C1-C6alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, (C1-C6 alkoxy)carbonyl,(C1-C6 alkyl)aminocarbonyl, di-(C1-C6 alkyl)aminocarbonyl, arylcarbonyl,aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, and arylsulfonyl.

and wherein R₂ is selected from the following: hydrogen, halo, C1-C6alkyl, tert-butyl, C2-C6 alkenyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6haloalkoxy, C2-C6 alkynyl, C3-C8 cycloalkenyl, (C3-C8 cycloalkyl)C1-C6alkyl, (C3-C8 cycloalkyl)C2-C6 alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy,C3-C7 heterocycloalkyl, (C3-C7 heterocycloalkyl)C1-C6 alkyl, (C3-C7heterocycloalkyl)C2-C6 alkenyl, (C3-C7 heterocycloalkyl)C1-C6 alkoxyl,hydroxy, carboxy, oxo, sulfanyl, C1-C6 alkyl furan, C2-C6 alkenyl furan,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, arylsulfonyl, and 2-acetyl-3-oxobutanimidoyl cyanide.

In some embodiments, each R₁ together from a C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring system. In one such embodiment, each R, togetherform benzene. In some of these embodiments, R₂ is tert-butyl. In otherof these embodiments, R₂ is 2-acetyl-3-oxobutanimidoyl cyanide (FormulaII). In some other of these embodiments, R₂ is a C1-C6 alkyl furan, suchas 2-propylfuran (Formula III), or a C2-C6 alkenyl furan, such as(E)-2-(prop-1-en-1-yl)furan (Formula IV).

In an embodiment, each R₁ is hydrogen. In some of these embodiments, R₂is tert-butyl. In other of these embodiments, R₂ is 3-oxobutanimidoylcyanide having a structure according to formula II. In some other ofthese embodiments, R₂ is a C1-C6 alkyl furan, such as 2-propylfuran, ora C2-C6 alkenyl furan, such as (E)-2-(prop-1-en-1-yl) furan.

The aPKC inhibitor or derivative thereof can exist as a salt or alcoholthereof, depending on protonation of the core compound as shown below:

PHARMACEUTICAL FORMULATIONS

Also provided herein are pharmaceutical formulations that contain aneffective amount of an aPKC inhibitor and/or derivative thereofdescribed herein and a pharmaceutically acceptable carrier appropriatefor administration to an individual in need thereof. In someembodiments, the aPKC inhibitor or derivative thereof is in the form ofa pharmaceutically acceptable salt. Suitable pharmaceutically acceptablesalts include, but are not limited to, sulfate, citrate, acetate,oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide,nitrate, bisulfate, phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate,napthalenesulfonate, propionate, malonate, mandelate, malate, phthalate,and pamoate. The pharmaceutical formulations or salts thereof can beadministered to a subject in need thereof. In some embodiments, thesubject has an aPKC abnormality. In further embodiments, the compoundsdescribed herein are used in the manufacture of a medicament for thetreatment of an aPKC abnormality.

Pharmaceutically Acceptable Carriers and Auxiliary Ingredients andAgents

The pharmaceutical formulations containing an effective amount of anaPKC inhibitor and/or derivative thereof further include apharmaceutically acceptable carrier. Suitable pharmaceuticallyacceptable carriers include, but are not limited to water, saltsolutions, alcohols, gum arabic, vegetable oils, benzyl alcohols,polyethylene glycols, gelatin, carbohydrates such as lactose, amylose orstarch, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinylpyrrolidone, which do not deleteriously react with the activecomposition.

The pharmaceutical formulations can be sterilized, and if desired, mixedwith auxiliary agents, such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, flavoring and/or aromatic substances, and the likewhich do not deleteriously react with the active composition.

In addition to the effective amount of an aPKC inhibitor and/orderivative thereof, the pharmaceutical formulations can also include aneffective amount of active agents, including but not limited to,antisense or RNA interference molecules, traditional chemotherapeutics,antineoplasic agents, immunomodulating compounds, hormones, antibiotics,antivirals, and/or antibodies or fragments thereof.

Effective Amounts of the aPKC Inhibitor, Derivative Thereof, andAuxiliary Active Agents

The effective amount of the aPKC inhibitor or derivative thereofcontained in the pharmaceutical formulation can range from about 0.001micrograms to about 1000 grams. In some embodiments, the effectiveamount ranges of the aPKC inhibitor or derivative thereof from about0.001 micrograms to about 0.01 micrograms. In other embodiments, theeffective amount of the aPKC inhibitor or derivative thereof ranges fromabout 0.01 micrograms to about 0.1 micrograms. In further embodiments,the effective amount of the aPKC inhibitor or derivative thereof rangesfrom about 0.1 micrograms to about 1.0 grams. In yet furtherembodiments, the effective amount of the aPKC inhibitor or derivativethereof ranges from about 1.0 grams to about 10 grams. In otherembodiments, the effective amount of the aPKC inhibitor or derivativethereof ranges from about 10 grams to about 100 grams. In still otherembodiments, the effective amount of the aPKC inhibitor or derivativethereof ranges from about 100 grams to about 1000 grams.

In embodiments where there is an auxiliary active agent contained in theaPKC inhibitor or derivative thereof pharmaceutical formulation, theeffective amount of the auxiliary active agent will vary depending onthe auxiliary active agent. In some embodiments, the effective amount ofthe auxiliary active agent ranges from 0.001 micrograms to about 1000grams. In other embodiments, the effective amount of the auxiliaryactive agent ranges from about 0.01 IU to about 1000 IU. In furtherembodiments, the effective amount of the auxiliary active agent rangesfrom 0.001 mL to about 1000 mL. In yet other embodiments, the effectiveamount of the auxiliary active agent ranges from about 1% w/w to about50% w/w of the total pharmaceutical formulation. In additionalembodiments, the effective amount of the auxiliary active agent rangesfrom about 1% v/v to about 50% v/v of the total pharmaceuticalformulation. In still other embodiments, the effective amount of theauxiliary active agent ranges from about 1% w/v to about 50% w/v of thetotal pharmaceutical formulation.

The auxiliary active agent can be included in the pharmaceuticalformulation or can exist as a stand-alone compound or pharmaceuticalformulation that is administered contemporaneously or sequentially withthe aPKC inhibitor, derivative thereof or pharmaceutical formulationthereof. In embodiments where the auxiliary active agent is astand-alone compound or pharmaceutical formulation, the effective amountof the auxiliary active agent can vary depending on the auxiliary activeagent used. In some of these embodiments, the effective amount of theauxiliary active agent ranges from 0.001 micrograms to about 1000 grams.In other embodiments, the effective amount of the auxiliary active agentranges from about 0.01 IU to about 1000 IU. In further embodiments, theeffective amount of the auxiliary active agent ranges from 0.001 mL toabout 1000 mL. In yet other embodiments, the effective amount of theauxiliary active agent ranges from about 1% w/w to about 50% w/w of thetotal auxiliary active agent pharmaceutical formulation. In additionalembodiments, the effective amount of the auxiliary active agent rangesfrom about 1% v/v to about 50% v/v of the total pharmaceuticalformulation. In still other embodiments, the effective amount of theauxiliary active agent ranges from about 1% w/v to about 50% w/v of thetotal auxiliary agent pharmaceutical formulation.

Dosage Forms

In some embodiments, the pharmaceutical formulations described hereinmay be in a dosage form. The dosage forms can be adapted foradministration by any appropriate route. Appropriate routes include, butare not limited to, oral (including buccal or sublingual), rectal,intraocular, inhaled, intranasal, topical (including buccal, sublingual,or transdermal), vaginal, parenteral, subcutaneous, intramuscular,intravenous, and intradermal. Such formulations may be prepared by anymethod known in the art.

Dosage forms adapted for oral administration can discrete dosage unitssuch as capsules, pellets or tablets, powders or granules, solutions, orsuspensions in aqueous or non-aqueous liquids; edible foams or whips, orin oil-in-water liquid emulsions or water-in-oil liquid emulsions. Insome embodiments, the pharmaceutical formulations adapted for oraladministration also include one or more agents which flavor, preserve,color, or help disperse the pharmaceutical formulation. Dosage formsprepared for oral administration can also be in the form of a liquidsolution that can be delivered as a foam, spray, or liquid solution. Theoral dosage form can be administered to a subject in need thereof. Insome embodiments, this is a subject having an aPKC abnormality.

Where appropriate, the dosage forms described herein can bemicroencapsulated.

The dosage form can also be prepared to prolong or sustain the releaseof any ingredient. In some embodiments, the aPKC inhibitor is theingredient whose release is delayed. In other embodiments, the releaseof an auxiliary ingredient is delayed. Suitable methods for delaying therelease of an ingredient include, but are not limited to, coating orembedding the ingredients in material in polymers, wax, gels, and thelike. Delayed release dosage formulations can be prepared as describedin standard references such as “Pharmaceutical dosage form tablets,”eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989),“Remington—The science and practice of pharmacy”, 20th ed., LippincottWilliams & Wilkins, Baltimore, Md., 2000, and “Pharmaceutical dosageforms and drug delivery systems”, 6th Edition, Ansel et al., (Media,Pa.: Williams and Wilkins, 1995). These references provide informationon excipients, materials, equipment, and processes for preparing tabletsand capsules and delayed release dosage forms of tablets and pellets,capsules, and granules. The delayed release can be anywhere from aboutan hour to about 3 months or more.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate, and hydroxypropyl methylcellulose acetate succinate;polyvinyl acetate phthalate, acrylic acid polymers and copolymers, andmethacrylic resins that are commercially available under the trade nameEUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, andpolysaccharides

Coatings may be formed with a different ratio of water soluble polymer,water insoluble polymers, and/or pH dependent polymers, with or withoutwater insoluble/water soluble non polymeric excipient, to produce thedesired release profile. The coating is either performed on the dosageform (matrix or simple) which includes, but is not limited to, tablets(compressed with or without coated beads), capsules (with or withoutcoated beads), beads, particle compositions, “ingredient as is”formulated as, but not limited to, suspension form or as a sprinkledosage form.

Where appropriate, the dosage forms described herein can be a liposome.In these embodiments, the aPKC inhibitor, derivative thereof, auxiliaryactive ingredient, and/or pharmaceutically acceptable salt thereof areincorporated into a liposome. In some embodiments, an aPKC inhibitor,derivative thereof, auxiliary active ingredient, and/or pharmaceuticallyacceptable salts thereof is integrated into the lipid membrane of theliposome. In other embodiments, an aPKC inhibitor, derivative thereof,auxiliary active ingredient, and/or pharmaceutically acceptable saltthereof are contained in the aqueous phase of the liposome. Inembodiments where the dosage form is a liposome, the pharmaceuticalformulation is thus a liposomal formulation. The liposomal formulationcan be administered to a subject in need thereof. In some embodiments,this is a subject having an aPKC abnormality.

Dosage forms adapted for topical administration can be formulated asointments, creams, suspensions, lotions, powders, solutions, pastes,gels, sprays, aerosols, or oils. In some embodiments for treatments ofthe eye or other external tissues, for example the mouth or the skin,the pharmaceutical formulations are applied as a topical ointment orcream. When formulated in an ointment, the aPKC inhibitor, derivativethereof, auxiliary active ingredient, and/or pharmaceutically acceptablesalt thereof can be formulated with a parafinnic or water-misicibleointment base. In other embodiments, the active ingredient can beformulated in a cream with an oil-in-water cream base or a water-in-oilbase. Dosage forms adapted for topical administration in the mouthinclude lozenges, pastilles, and mouth washes.

Dosage forms adapted for nasal or inhalation administration includeaerosols, solutions, suspension drops, gels, or dry powders. In someembodiments, the aPKC inhibitor, derivative thereof, auxiliary activeingredient, and/or pharmaceutically acceptable salt thereof in a dosageform adapted for inhalation is in a particle-size-reduced form that isobtained or obtainable by micronization. In some embodiments, theparticle size of the size reduced (e.g. micronized) compound or salt orsolvate thereof, is defined by a D50 value of about 0.5 to about 10microns as measured by an appropriate method known in the art. Dosageforms adapted for administration by inhalation also include particledusts or mists. Suitable dosage forms wherein the carrier or excipientis a liquid for administration as a nasal spray or drops include aqueousor oil solutions/suspensions of an active ingredient, which may begenerated by various types of metered dose pressurized aerosols,nebulizers, or insufflators.

In some embodiments, the dosage forms are aerosol formulations suitablefor administration by inhalation. In some of these embodiments, theaerosol formulation contains a solution or fine suspension of an aPKCinhibitor, derivative thereof, auxiliary active ingredient, and/orpharmaceutically acceptable salt thereof a pharmaceutically acceptableaqueous or non-aqueous solvent. Aerosol formulations can be presented insingle or multi-dose quantities in sterile form in a sealed container.For some of these embodiments, the sealed container is a single dose ormulti-dose nasal or an aerosol dispenser fitted with a metering valve(e.g. metered dose inhaler), which is intended for disposal once thecontents of the container have been exhausted.

Where the aerosol dosage form is contained in an aerosol dispenser, thedispenser contains a suitable propellant under pressure, such ascompressed air, carbon dioxide, or an organic propellant, including butnot limited to a hydrofluorocarbon. The aerosol formulation dosage formsin other embodiments are contained in a pump-atomizer. The pressurizedaerosol formulation can also contain a solution or a suspension of anaPKC inhibitor, derivative thereof, auxiliary active ingredient, and/orpharmaceutically acceptable salt thereof. In further embodiments, theaerosol formulation also contains co-solvents and/or modifiersincorporated to improve, for example, the stability and/or taste and/orfine particle mass characteristics (amount and/or profile) of theformulation.

For some dosage forms suitable and/or adapted for inhaledadministration, the pharmaceutical formulation is a dry powder inhalableformulations. In addition to the aPKC inhibitor, derivative thereof,auxiliary active ingredient, and/or pharmaceutically acceptable saltthereof, such a dosage form can contain a powder base such as lactose,glucose, trehalose, manitol, and/or starch. In some of theseembodiments, the aPKC inhibitor, derivative thereof, auxiliary activeingredient, and/or pharmaceutically acceptable salt thereof is in aparticle-size reduced form. In further embodiments, a performancemodifier, such as L-leucine or another amino acid, cellobioseoctaacetate, and/or metals salts of stearic acid, such as magnesium orcalcium stearate.

In some embodiments, the aerosol formulations are arranged so that eachmetered dose of aerosol contains a predetermined amount of an activeingredient, such as the one or more of the compounds described herein.

Dosage forms adapted for vaginal administration can be presented aspessaries, tampons, creams, gels, pastes, foams, or spray formulations.Dosage forms adapted for rectal administration include suppositories orenemas.

Dosage forms adapted for parenteral administration and/or adapted forinjection can include aqueous and/or non-aqueous sterile injectionsolutions, which can contain anti-oxidants, buffers, bacteriostats,solutes that render the composition isotonic with the blood of thesubject, and aqueous and non-aqueous sterile suspensions, which caninclude suspending agents and thickening agents. The dosage formsadapted for parenteral administration can be presented in a single-unitdose or multi-unit dose containers, including but not limited to sealedampoules or vials. The doses can be lyophilized and resuspended in asterile carrier to reconstitute the dose prior to administration.Extemporaneous injection solutions and suspensions can be prepared insome embodiments, from sterile powders, granules, and tablets.

Methods of Making aPKC Inhibitors and Derivatives Thereof

The aPKC inhibitors and derivatives thereof can be synthesized via manymethods generally known to those skilled in the art. The presentdisclosure is not intended to be limited by the particular methods ofsynthesizing the aPKC inhibitors. For example, the core compound ofFormula 1, wherein R₁ are each hydrogen and R₂ is a methyl group, can besynthesized from acid anhydrides and isopropenyl acetate as described inMerenyi and Nilsson, 1964. Acta. Chem. Scan. 18:1368-1372. Other aPKCinhibitors and derivatives thereof may or may not be synthesizedstarting from the core compound of Formula I. The skilled artisan willrecognize additional methods of synthesizing the aPKC inhibitorsdescribed herein. For example, Muxfeldt, et al., 1968. J. Org. Chem.33(4):1645-1647 describes the synthesis of derivatives ofcyclopentane-1,3-dione from oxazolones; U.S. Pat. No. 3,381,035describes methods of synthesizing 2-substituted cyclopentane-1,3-diones;and U.S. Pat. No. 3,773,622 describes methods of synthesizing2-substituted-4-hydroxy-cyclopentane-1,3-diones.

Methods of Using aPKC Inhibitors and Formulations Thereof

Any amount, but particularly the effective amount of the aPKCinhibitors, derivatives thereof, and formulations thereof describedherein can be administered as a dose one or more times per day, week,month, or year. Multiple doses can be give contemporaneously, such as inthe case of an aerosol formulation, or can be givennon-contemporaneously, such as multiple times per day where theadministration is separated by at least about 2 minutes.

The effective amount of the aPKC inhibitor can be given in a singledose. In other embodiments, the effective amount of the aPKC inhibitorcompound or derivative thereof can be administered over multiple doses,in which each contains a fraction of the total effective amount to beadministered (“sub-doses”).

In some embodiments, the amount of doses and/or sub-doses delivered perday is 2, 3, 4, 5, or 6. In further embodiments, the doses or sub-dosesare administered one or more times per week, such as 1, 2, 3, 4, 5, or 6times per week. In other embodiments, the doses and/or sub-doses areadministered one or more times per month, such as 1 to 8 times permonth. In still further embodiments, doses and/or sub-doses one or moretimes per year, such as 1 to 11 times per year.

In some embodiments, an auxiliary active agent is used with an aPKCinhibitor, derivative thereof, or formulation thereof but is notdirectly included in the pharmaceutical formulation. In some of theseembodiments, the aPKC inhibitor, derivative thereof, or formulationthereof the auxiliary agent can be administered contemporaneously withthe aPKC inhibitor or derivative thereof. In one non-limiting example,during or at the time of oral administration of a dose or sub dose ofthe aPKC inhibitor formulation, the patient can receive an injection ofthe auxiliary agent. In another non-limiting example, the patient couldtake one tablet containing a dose of the aPKC inhibitor formulation andanother tablet containing a dose of the aPKC inhibitor. In otherembodiments, the auxiliary agent can be administered sequentially withthe aPKC inhibitor, derivative thereof, or formulation thereof, whereinthe administration of the aPKC inhibitor, derivative thereof, orformulation thereof and the auxiliary active agent is separated by anamount of time greater than about 10 minutes.

Kits Containing an aPKC Inhibitor or Formulations Thereof

The compounds and pharmaceutical formulations described herein can bepresented as a combination kit. As used herein, the terms “combinationkit” or “kit of parts,” or “kit” refers to the compound orpharmaceutical formulations and additional components that are used topackage, sell, market, deliver, and/or administer the combination ofelements or a single element, such as the active ingredient, containedtherein. Such additional components include but are not limited to,packaging, syringes, blister packages, bottles, and the like. When oneor more of the components (e.g. active agents) contained in the kit areadministered simultaneously, the combination kit can contain the activeagents in a single pharmaceutical formulation (e.g. a tablet) or inseparate pharmaceutical formulations.

In some embodiments, the aPKC inhibitor or derivative thereof is keptseparate from a diluent or other ingredient (active or inactive) untilimmediately prior to use. In these embodiments, the kit contains theaPKC inhibitor or derivative thereof in separate packaging from theother ingredient.

In embodiments where the aPKC inhibitor, derivative thereof, orpharmaceutical formulation thereof are administered along with aseparate auxiliary active agent, the combination kit can contain eachagent in separate pharmaceutical formulations. The separatepharmaceutical formulations (e.g. the aPKC inhibitor pharmaceuticalformulation and the auxiliary active agent formulation) can be containedin a single package or in separate packages within the kit.

In some embodiments, the combination kit also includes instructionsprinted on or otherwise contained in a tangible medium of expression.The instructions can provide information regarding the content of thecompound or pharmaceutical formulations contained therein, safetyinformation regarding the content of the compound(s) or pharmaceuticalformulation(s) contained therein, information regarding the dosages,indications for use, and/or recommended treatment regimen(s) for thecompound(s) and/or pharmaceutical formulations contained therein. Insome embodiments, the instructions provide directions for administeringthe compounds, pharmaceutical formulations, or salts thereof to asubject having an aPKC abnormality. In some embodiments, the aPKCabnormality is obesity, glucose intolerance, metabolic syndrome,hyperinsulinemia, hepatosteatosis, non-alcoholic cirrhosis,hypertriglyceridemia, hypercholesterolemia, polycystic ovary disease,and Alzheimer's disease.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure. References cited incorporated by reference as if expressedin their entirety.

Example 1 Potential aPKC Inhibitor Compounds

A computational approach was used to identify potential aPKC inhibitorcompounds. Some of these compounds are commercially available and otherscan be made using techniques known to the skilled artisan as describedabove. Briefly, a theoretical structure of PKC_(ι) was used to screencompounds from a database to identify compounds with structures that canfit in the binding pocket and thus bind aPKCs. A compound(2-acetylcyclopentane-1,3-dione (ACPD), Formula XI) having the generalcore structure described herein was identified and tested for itsability to bind and inhibit aPKC as described in the Examples herein.ACPD can be synthesized as described above or obtained commercially.2-acetylcyclopentane-1,3-dione (A CPD)

Other compounds were identified from the computational database searchthat contained the same core structure as ACPD, and are thus alsoexpected to be aPKC inhibitors. These additional compounds are asfollows:

-   2-(3-(furan-2-yl)propanoyl)cyclopentane-1,3-dione-   2-(3-(furan-2-yl)propanoyl)cyclopentane-1,3-dione is an example    potential aPKC inhibitor and is shown according to Formula V.

-   (E)-2-(3-(furan-2-yl)acryloyl)cyclopentane-1,3-dione-   (E)-2-(3-(furan-2-yl)acryloyl)cyclopentane-1,3-dione is an example    potential aPKC inhibitor and is shown according to Formula VI.

-   2-(3-(furan-2-yl)propanoyl)-1H-indene-1,3(2H)-dione-   2-(3-(furan-2-yl)propanoyl)-1H-indene-1,3(2H)-dione is an example    potential aPKC inhibitor and is shown according to Formula VII.

-   (E)-2-(3-(furan-2-yl)acryloyl)-1H-indene-1,3(2H)-dione-   (E)-2-(3-(furan-2-yl)acryloyl)-1H-indene-1,3(2H)-dione is an example    potential aPKC inhibitor and is shown according to Formula VIII.

-   2-pivaloyl-1H-indene-1, 3(2H)-dione and Synthesis Thereof-   2-pivaloyl-1H-indene-1,3(2H)-dione is an example potential aPKC    inhibitor and is shown according to Formula IX.

-   2-pivaloylcyclopentane-1,3-dione-   2-pivaloylcyclopentane-1,3-dione is an example potential aPKC    inhibitor and is shown according to Formula X.

Example 2 Dose-Related Effects of ACPD on PIP3-Stimulated Activities ofPKC-ι and PKC-ζ and PMA-Stimulated Activities of PKC-α, PKC-β2, PKC-δ,and PKC-ε

Methods

ACPD used in this Example was obtained from Sigma (St. Louis, Mo., USA).PIP3-stimulated activities of PKC-ι and PKC-ζ were measured as follows.10 ng of recombinant forms of human aPKCs (PKC-ι and PKC-ζ) (Biovision,Mountain, Calif., USA) were assayed for aPKC activity in the presence of10 fmol/l phosphatidylinositol-3,4,5-(PO₄)₃ (PIP₃) (Matreya, PleasantGap, Pa., USA) with and without addition of varying amounts of ACPD.PIP₃ was added to activate and define aPKC activity. The aPKC activityassay was performed as described in (for example) Sajan et al. 2012.Metabolism. 61:459-469. Briefly, recombinant aPKCs were incubated for 8minutes at 30° C. in 100 μl buffer containing 50 mmol/l Tris/HCl (pH,7.5), 100 μmol/l Na₃VO₄, 100 μmol/l Na₄P₂O₄, 1 mmol/l NaF, 100 μmol/lPMSF, 4 μg phosphatidylserine (Sigma, St. Louis, Mo., USA), 50 μmol/l[γ-³²PO₄]ATP (NEN Life Science Products, Beverly, Mass., USA), 5 mmol/lMgCl₂, and, as substrate, 40 mol/l serine analogue of the PKC-εpseudosubstrate (Millipore, Bedford, Mass., USA). After incubation,³²P-labeled substrate was trapped on P-81 filter paper and counted. Thisassay reflects the specific activity of a constant amount of the aPKCs.

Additionally, recombinant forms of human PKC-α, PKC-β2, PKC-δ andPKC-ε(gifts from Sphinx Division, Lilly Corp., Indianapolis, Ind., USA)were assayed for their activity, as previously described with respect tothe recombinant aPKCs, in the presence of 1 mmol/l CaCl2 and 100 nmol/lphorbol myristoyl acetate (PMA) for PKC-α and PKC-β2, and in thepresence of 100 nmol/l PMA for PKC-δ and PKC-ε to activate and definerespective PKC activities. PMA was added to activate and define the PKCactivity.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

Results from the aPKC and PKC activity assays are shown in FIGS. 1 and2. FIG. 1 shows the dose-related effects of ACPD on PIP3-stimulatedactivities of PKC-ι (FIG. 1A) and PKC-ζ(FIG. 1B). Values are mean±SEM of4 determinations. FIG. 2 shows dose-related effects of ACPD on PhorbolMyristoyl Acetate (PMA)-±CaCl₂-stimulated Activities of PKC-α (FIG. 2A),PKC-β2 (FIG. 2B), PKC-δ (FIG. 2C) and PKC-ε (FIG. 2D). As shown in FIG.1, ACPD reduces the activities of PKC-ι (FIG. 1A) and PKC-ζ (FIG. 1B) ina dose-dependent manner. ACPD did not reduce activities of PKC-α (FIG.2A), PKC-β2 (FIG. 2B), PKC-δ (FIG. 2C) and PKC-ε (FIG. 2D). As shown inFIG. 1, ACPD had comparable potencies (approximately Ki, 10⁻⁷ mol/l) fordiminishing the activities of both PKC-ζ and PKC-ι. Moreover, ACPD waswithout effect on activities of recombinant forms of PKC-α, PKC-β2,PKC-δ and PKC-ε stimulated maximally with phorbol±calcium chloride (FIG.2), suggesting that ACPD is an aPKC-specific, pan-aPKC inhibitor,meaning that ACPD inhibits both forms of aPKC enzymes without having aneffect on the activity of other PKC enzymes.

Example 3 Dose-Related Effects of ACPD on Activities of Total aPKC andAkt2 in Control and Insulin-Stimulated Hepatocytes of Non-DiabeticHumans

Methods

Hepatocyte Incubations:

Cryo-preserved hepatocytes (70-90% viability; purchased from Zen-BioCorp, Research Triangle, NC, USA, were harvested from perfused livers ofnon-diabetic subjects [2 females and 6 males; ages, 43-60 years, 51±3(mean±SEM); BMI, 30±2] and type 2 diabetic subjects [2 females and 4males, ages, 46-68 years, 60±4; BMI, 27±2] maintained on life support astransplant donors (these hepatocytes were obtained from the same patientgroups described in Sajan and Farese. 2012. Diabetologia. 55:1446-1457).Diabetic subjects were hyperglycaemic and undergoing insulin treatment.Other pertinent laboratory and clinical data are not available intransplant donors.

Hepatocytes were incubated (about 10⁶ cells/100 mm plate) overnight(approx. 16 hours) in Dulbecco's minimal essential medium containingabout 5% fetal calf serum, about 100 units/ml sodium-penicillin, about100 μg/ml streptomycin-sulfate, about 2 μmol/l dexamethasone, followedby incubation for about 2 hours in William's E medium (Sigma, St. Louis,Mo., USA) containing Glutamax (Invitrogen, Carlsbad, Calif., USA),100units/ml sodium-penicillin, 100 μg/ml streptomycin-sulfate, 100 nmol/ldexamethasone, followed by incubation for about 4 hours in similarmedium supplemented with about 25 mg/ml transferrin and about 0.25 μg/mlsodium selenite. Where indicated, about 1 μmol/l insulin and varyingconcentrations of ACPD were present in the media throughout allincubations. This concentration of insulin was added to maintain a highlevel of insulin activation of aPKC during prolonged incubation. Forcomparison, about 100 nmol/l insulin was less effective than about 1μmol/l insulin in maintaining increases in aPKC and Akt activity innon-diabetic hepatocytes.

All experimental procedures involving human materials were approved bythe Institutional Review Board of the University of South FloridaCollege of Medicine and the James A. Haley Veterans AdministrationMedical Center Research and Development Committee, Tampa, Fla., andconducted in accordance with the Declaration of Helsinki and GoodClinical Practice.

Activity Assays:

As described in Sajan and Farese. 2012. Diabetologia. 55:1446-1457,aPKCs were immunopercipitated from lysates with rabbit polyclonalantiserum (Santa Cruz Biotechnologies, Santa Cruz, Calif., USA) whichrecognizes C-termini of PKC-ζ and PKC-λ/ι. Note that PKC-ι is the humanhomolog of mouse PKC-ζ with 98% homology; human and mouse muscle containprimarily PKC-ι/λ and little PKC-ζ; mouse and human liver containsubstantial amounts of both PKC-ι/λ and PKC-ζ(Farese and Sajan. 2010.Am. J. Physiol. Endocrinol. Metab. 298:E385-394).

To measure aPKC activity, immunoprecipitates were collected onSepharose-AG beads (Santa Cruz Biotechnologies) and incubated for 8minutes at 30° C. in 100 μl buffer containing 50 mmol/l Tris/HCl (pH,7.5), 100 μmol/l Na₃VO₄, 100 μmol/l Na₄P₂O₄, 1 mmol/l NaF, 100 μmol/lPMSF, 4 μg phosphatidylserine (Sigma, St. Louis, Mo., USA), 50 μmol/l[γ-³²PO₄]ATP (NEN Life Science Products, Beverly, Mass., USA), 5 mmol/lMgCl₂, and, as substrate, 40 μmol/l serine analogue of the PKC-εpseudosubstrate (Millipore, Bedford, Mass., USA). After incubation,³²P-labeled substrate was trapped on P-81 filter paper and counted. aPKCactivation was also assessed by immunoblotting for phosphorylation ofthe auto(trans)phosphorylation site, thr-555/560 in PKC-ι/4, requiredfor, and reflective of, activation (Farese and Sajan. 2010. Am. J.Physiol. Endocrinol. Metab. 298:E385-394).

Akt2 enzyme activity was assayed in immunoprecipitates using a kitpurchased from Millipore, as described in Sanjan et. al. 2010. Am. J.Physiol. Endocrinol. Metab. 298:E179-192 and Sajan and Farese (2012).Diabetologia. 55:1446-1457. Akt activity was also assessed byimmunoblotting for phosphorylation of ser-473-Akt.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

As shown in FIG. 3A, ACPD inhibited insulin-stimulated increases intotal aPKC activity in hepatocytes of non-diabetic humans, withhalf-maximal and maximal inhibition occurring at approximately 10⁻⁷ and10⁻⁶ mol/ml. The concentrations of ACPD required for inhibition of totalaPKC in human hepatocytes were similar to those required for inhibitionof recombinant forms of PKC-ι and PKC-ζ. Basal activity (FIG. 3A, openbars), on the other hand, was not affected by ACPD, suggesting thateither the activated form of aPKC is more open and accessible forinteraction with inhibitor or non-aPKC kinases co-immunoprecipitate withaPKC and are not inhibited by ACPD. In any case, it is only thestimulated activity that can more clearly be attributed to aPKC. Asshown in FIG. 3B, ACPD did not inhibit basal (open bars) orinsulin-stimulated increases in hepatic Akt2 activity. Similarly, ACPDhad no effects on activity of AMP-activated protein kinase (AMPK).

Example 4 Effect of ACPD on Expression of Lipogenic and GluconeogenicFactors in Basal and Insulin-Stimulated Hepatocytes of Non-Diabetic andType 2 Diabetic (T2DM) Humans

Methods

Hepatocyte Incubations:

Cryo-preserved hepatocytes (70-90% viability; purchased from Zen-BioCorp, Research Triangle, NC, USA, were harvested from perfused livers ofnon-diabetic subjects [2 females and 6 males; ages, 43-60 years, 51±3(mean±SEM); BMI, 30±2] and type 2 diabetic subjects [2 females and 4males, ages, 46-68 years, 60±4; BMI, 27±2] maintained on life support astransplant donors (these hepatocytes were obtained from the same patientgroups described in Sajan and Farese. 2012. Diabetologia. 55:1446-1457).Diabetic subjects were hyperglycaemic and undergoing insulin treatment.Other pertinent laboratory and clinical data are not available intransplant donors.

Hepatocytes were incubated (about 10⁶ cells/100 mm plate) overnight(approx. 16 hours) in Dulbecco's minimal essential medium containingabout 5% fetal calf serum, about 100 units/ml sodium-penicillin, about100 μg/ml streptomycin-sulfate, about 2 μmol/l dexamethasone, followedby incubation for about 2 hours in William's E medium (Sigma, St. Louis,Mo., USA) containing Glutamax (Invitrogen, Carlsbad, Calif., USA),100units/ml sodium-penicillin, 100 μg/ml streptomycin-sulfate, 100 nmol/ldexamethasone, followed by incubation for about 4 hours in similarmedium supplemented with about 25 mg/ml transferrin and about 0.25 μg/mlsodium selenite. Where indicated, about 1 μmol/l insulin and varyingconcentrations of ACPD were present in the media throughout allincubations. This concentration of insulin was added to maintain a highlevel of insulin activation of aPKC during prolonged incubation. Forcomparison, about 100 nmol/l insulin was less effective than about 1μmol/l insulin in maintaining increases in aPKC and Akt activity innon-diabetic hepatocytes.

All experimental procedures involving human materials were approved bythe Institutional Review Board of the University of South FloridaCollege of Medicine and the James A. Haley Veterans AdministrationMedical Center Research and Development Committee, Tampa, Fla., andconducted in accordance with the Declaration of Helsinki and GoodClinical Practice.

Cell Preparation:

The treated hepatocytes were homogenized in ice-cold buffer containing0.25 mol/l sucrose, 20 mmol/l Tris/HCl (pH, 7.5), 2 mmol/l EGTA, 2mmol/l EDTA, 1 mmol/l phenlysulfonlyfluoride (PMSF), 20 μg/ml leupeptin,10 μg/ml aprotinin, 2 mmol/l Na4P2O7, 2 mmol/l Na3VO4, 2 mmol/l NaF, and1 μmol/l microcystin, and then supplemented with 1% TritonX-100, 0.6%Nonidet and 150 mmol/l NaCl, and cleared by low-speed centrifugation.

mRNA Quantification:

Cell preparations were added to Trizol reagent (Invitrogen) and RNA wasextracted and purified with RNA-Easy Mini-Kit and RNAase-free DNAase Set(Qiagen, Valencia, Calif., USA), quantified (A₂₆₀/A₂₈₀), checked forpurity by electrophoresis on 1.2% agarose gels, and quantified byquantitative real-time reverse transcriptase-polymerase chain reaction(RT-PCR), using TaqMan reverse transcription reagent and SYBR Green kit(Applied Biosystems, Carlsbad, Calif., USA) with human nucleotideprimers shown in Table 1 and as described in Sajan et al. 2012.Metabolism. 61:459-469. mRNA expression is normalized to hypoxanthinephosphoribosyl-transferase (HPT) mRNA expression.

TABLE 1 GENE Forward Primer Sequence (5′→3′) Reverse Primer Sequence(5′→3′) SREBP 1 SEQ ID NO: 1 SEQ ID NO: 2 ATCGGCGCGGAAGCTGTCGGGGTAGACTGTCTTGGTTGATGAGCTGGAGC CGTC AT PEPCK SEQ ID NO: 3 SEQ ID NO: 4GACAGCCTGCCCCAGGCAGTGA CTGGCCACATCTCGAGGGTCAG FAS SEQ ID NO: 5 SEQ IDNO: 6 ACCGACTTCATGAATTTGCTGAT AAGCTGAAAGCTTTCTGTCT G6Pase SEQ ID NO: 7SEQ ID NO: 8 TGCTGCTCACTTTCCCCACCAG TCTCCAAAGTCCACAGGAGGT HPT SEQ ID NO:9 SEQ ID NO: 10 TGAAAGACTTGCTCGAGATGT AAAGAACTTATAGCCCCCCTT

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

As demonstrated in FIG. 4, ACPD diminished insulin-dependent increasesin expression of lipogenic factors, SREBP-1c (Figure A) and FAS (FIG.4C), in hepatocytes of non-diabetic humans. ACPD also diminisheddiabetes-dependent increases in SREBP-1c and FAS in hepatocytes of type2 diabetic humans (FIGS. 4A and 4C, respectively). Insulin did notincrease SREBP-1c and FAS mRNA beyond the increases provoked by type 2diabetes. This suggests that aPKC activity is already maximally or nearmaximally activated in diabetic hepatocytes and consequently unable torespond to further stimulation.

ACPD diminished basal expression of PEPCK and G6Pase in non-diabetichepatocytes (FIGS. 4B and 4D, respectively). ACPD also diminisheddiabetes-dependent increases in PEPCK and G6Pase expression inhepatocytes of type 2 diabetic humans (FIGS. 4B and 4D, respectively).Insulin diminished the expression of PEPCK and G6Pase in non-diabetichepatocytes, but not in hepatocytes of type 2 diabetic humans (FIGS. 4Band 4D, respectively). This suggests diminished activation of Akt byinsulin is seen in these hepatocytes or uncoupling of Akt fromdownstream factors that directly control gluconeogenic genetranscription, such as FoxO1, or both. See e.g. FIG. 24.

Example 5 Effect of ACPD on Resting/Basal and Insulin-Stimulated aPKCActivity in Mouse Liver and Muscle

Methods

Standard (C57Bl/6/SV129) mice consuming standard low-fat mouse chow wereinjected subcutaneously (s.c.) with a single injection of ACPD (10 mg/kgbody weight). At about 0, 24, 48, and 72 hours post injection, mice weretreated with or without an intraperitoneal injection of insulin (1 U/kgbody weight) or control 15 minutes prior to killing. Post mortem, liverand muscle tissues were removed and examined for immunoprecipitable aPKCactivity as described below.

Tissue Preparation:

liver and muscle tissue samples were homogenized in an ice-cold buffercontaining 0.25 mol/l sucrose, 20 mmol/l Tris/HCl (pH, 7.5), 2 mmol/lEGTA, 2 mmol/l EDTA, 1 mmol/l phenlysulfonlyfluoride (PMSF), 20 μg/mlleupeptin, 10 μg/ml aprotinin, 2 mmol/l Na4P207, 2 mmol/l Na3VO4, 2mmol/l NaF, and 1 μmol/l microcystin, and then supplemented with 1%TritonX-100, 0.6% Nonidet and 150 mmol/l NaCl, and cleared by low-speedcentrifugation.

aPKC Activity Assay:

As described in Sajan and Farese. 2012. Diabetologia. 55:1446-1457,aPKCs were immunopercipitated from lysates with rabbit polyclonalantiserum (Santa Cruz Biotechnologies, Santa Cruz, Calif., USA) whichrecognizes C-termini of PKC-□ and PKC-λ/ι. Note that PKC-ι is the humanhomolog of mouse PKC-ζ with 98% homology; human and mouse muscle containprimarily PKC-ι/λ and little PKC-ζ; mouse and human liver containsubstantial amounts of both PKC-ι/λ and PKC-ζ(Farese and Sajan. 2010.Am. J. Physiol. Endocrinol. Metab. 298:E385-394).

To measure aPKC activity, immunoprecipitates were collected onSepharose-AG beads (Santa Cruz Biotechnologies) and incubated for 8minutes at 30° C. in 100 μl buffer containing 50 mmol/l Tris/HCl (pH,7.5), 100 μmol/l Na₃VO₄, 100 μmol/l Na₄P₂O₄, 1 mmol/l NaF, 100 μmol/lPMSF, 4 μg phosphatidylserine (Sigma, St. Louis, Mo., USA), 50 μmol/l[γ-³²PO₄]ATP (NEN Life Science Products, Beverly, Mass., USA), 5 mmol/lMgCl₂, and, as substrate, 40 μmol/l serine analogue of the PKC-εpseudosubstrate (Millipore, Bedford, Mass., USA). After incubation,³²P-labeled substrate was trapped on P-81 filter paper and counted. aPKCactivation was also assessed by immunoblotting for phosphorylation ofthe auto(trans)phosphorylation site, thr-555/560 in PKC-ι/4, requiredfor, and reflective of, activation (Farese and Sajan. 2010. Am. J.Physiol. Endocrinol. Metab. 298:E385-394).

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

As shown in FIG. 5, treatment of normal mice with a single s.c. dose ofACPD (10 mg/kg body weight) elicited a decrease in hepaticinsulin-stimulated aPKC activity that was observed at 24 hours post ACPDtreatment and returned to pre-treatment levels over the following 48hours (FIG. 5A). In contrast, insulin-stimulated aPKC activity in musclewas not altered by ACPD treatment at any time point (FIG. 5B), Thissuggests that in normal, low-fat fed mice and as administered in thisExample, ACPD selectively inhibited hepatic aPKC.

Example 6 Shows the Effect of High Fat Feeding (HFF) and ACPD or ICAP(s.c. Injection of ICAP, 1 mg/kg Body Weight, or ACPD; 10 mg/kg BodyWeight) on Basal and Insulin-Stimulated Activity of aPKC and Akt2 inMuscle and Liver

Methods

Standard (C57Bl/6/SV129) mice consuming standard low-fat mouse chow wereinjected subcutaneously (s.c.) with a daily single injection of ICAP (1mg/kg body weight) or ACPD (10 mg/kg body weight). After 10 weeks offeeding a low fat diet (10% calories from fat) (control mice) or a highfat diet (40% of calories from milk fat) and daily treatment with orwithout aPKC inhibitor (ACPD or 1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphono-oxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)(ICAP)), mice were injected with or without insulin (1 U/kg body weightgiven intraperitoneally) and killed 15 minutes post insulin injection.Post mortem, liver and muscle tissues were removed and examined forimmunoprecipitable aPKC activity and akt2 as described below.

Tissue Preparation: Liver and muscle tissue samples were homogenized inice-cold buffer containing 0.25 mol/l sucrose, 20 mmol/l Tris/HCl (pH,7.5), 2 mmol/l EGTA, 2 mmol/l EDTA, 1 mmol/l phenlysulfonlyfluoride(PMSF), 20 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mmol/l Na4P207, 2mmol/l Na3VO4, 2 mmol/l NaF, and 1 μmol/l microcystin, and thensupplemented with 1% TritonX-100, 0.6% Nonidet and 150 mmol/l NaCl, andcleared by low-speed centrifugation.

aPKC Activity Assay:

As described in Sajan and Farese. 2012. Diabetologia. 55:1446-1457,aPKCs were immunopercipitated from lysates with rabbit polyclonalantiserum (Santa Cruz Biotechnologies, Santa Cruz, Calif., USA) whichrecognizes C-termini of PKC-ζ and PKC-λ/ι. Note that PKC-ι is the humanhomolog of mouse PKC-ζ with 98% homology; human and mouse muscle containprimarily PKC-ι/λ and little PKC-ζ; mouse and human liver containsubstantial amounts of both PKC-ι/λ and PKC-ζ(Farese and Sajan. 2010.Am. J. Physiol. Endocrinol. Metab. 298:E385-394).

To measure aPKC activity, immunoprecipitates were collected onSepharose-AG beads (Santa Cruz Biotechnologies) and incubated for 8minutes at 30° C. in 100 μl buffer containing 50 mmol/l Tris/HCl (pH,7.5), 100 μmol/l Na₃VO₄, 100 μmol/l Na₄P₂O₄, 1 mmol/l NaF, 100 μmol/lPMSF, 4 μg phosphatidylserine (Sigma, St. Louis, Mo., USA), 50 μmol/l[γ-³²PO₄]ATP (NEN Life Science Products, Beverly, Mass., USA), 5 mmol/lMgCl₂, and, as substrate, 40 μmol/l serine analogue of the PKC-εpseudosubstrate (Millipore, Bedford, Mass., USA). After incubation,³²P-labeled substrate was trapped on P-81 filter paper and counted. aPKCactivation was also assessed by immunoblotting for phosphorylation ofthe auto(trans)phosphorylation site, thr-555/560 in PKC-ι/4, requiredfor, and reflective of, activation (Farese and Sajan. 2010. Am. J.Physiol. Endocrinol. Metab. 298:E385-394).

Akt2 enzyme activity was assayed in immunoprecipitates using a kitpurchased from Millipore, as described in Sanjan et. al. 2010. Am. J.Physiol. Endocrinol. Metab. 298:E179-192 and Sajan and Farese (2012).Diabetologia. 55:1446-1457.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

In control mice consuming a standard chow, low fat diet (10% caloriesfrom fat), insulin increased phosphorylation and enzyme activity of bothaPKC and Akt2 in liver and muscle (FIG. 6). However, in HFF mice basalhepatic activities of both aPKC and Akt2 were elevated (FIGS. 6A and6C). This most likely reflects hyperinsulinemia in these mice. For aPKC,this suggests increases in lipid activators of aPKC in these mice.Insulin appeared to have provoked further increases in the activities ofhepatic aPKC and Akt2 to levels comparable to or exceeding thoseattained in control mice (FIGS. 6A and 6C). These data suggest thatinsulin signaling to both hepatic factors was fully intact in this modelof modestly increased dietary fat.

Treatment of HFF mice with the doses of ICAP (1 mg/kg body-weight) andACPD (10 mg/kg body-weight) diminished resting and insulin-stimulatedhepatic aPKC activity to control levels (FIG. 6A). On the other hand,hepatic Akt2 activity, if anything, increased further with ICAP and ACPDtreatment (FIG. 6C). These data suggest that both inhibitorsspecifically inhibited aPKC and at least spared Akt2 and may haveenhanced Akt2 activation. ICAP is specific for PKC-ι/λ, whereas ACPD isspecific for both PKC-ι/λ and PKC-ζ. Neither ICAP nor ACPD affects otherPKCs.

In contrast to liver, basal, and insulin-stimulated phosphorylation,activities of both aPKC and Akt2 were diminished in muscles of HFF mice(FIGS. 6B and 6D, respectively).

Moreover, in muscle, treatment with ICAP or ACPD increased both aPKC andAkt2 activity toward, albeit not fully to, normal levels (FIGS. 6A and6B). The reason for the hepatic selectivity of both ICAP and ACPD isbeneficial for simultaneously improving muscle and liver.

Example 7 Effects of HFF Diet and ICAP (I) or ACPD (A) on HepaticResting/Basal and Insulin Stimulated Phosphorylation of Ser⁴⁷³-Akt,Ser9-GSK3β, Thr^(555/560)-PKC-λ/ι, and Thr2⁴⁴⁸-mTOR

Methods

Standard (C57Bl/6/SV129) mice consuming standard low-fat mouse chow wereinjected subcutaneously (s.c.) with a daily single injection of ICAP (1mg/kg body weight) or ACPD (10 mg/kg body weight). After 10 weeks offeeding a low fat diet (10% calories from fat) (control mice) or a highfat diet (40% of calories from milk fat) and daily treatment with orwithout aPKC inhibitor (ACPD or 1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphono-oxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)(ICAP)), mice were injected with or without insulin (1 U/kg body weightgiven intraperitoneally) and killed 15 minutes post insulin injection.Post mortem, liver tissue was removed and examined for immunoreactivityof the indicated signaling factors.

Tissue Preparation:

To prepare cell lysates, liver and muscle tissue samples werehomogenized in ice-cold buffer containing 0.25 mol/l sucrose, 20 mmol/lTris/HCl (pH, 7.5), 2 mmol/l EGTA, 2 mmol/l EDTA, 1 mmol/lphenlysulfonlyfluoride (PMSF), 20 μg/ml leupeptin, μg/ml aprotinin, 2mmol/l Na4P207, 2 mmol/l Na3VO4, 2 mmol/l NaF, and 1 μmol/l microcystin,and then supplemented with 1% TritonX-100, 0.6% Nonidet and 150 mmol/lNaCl, and cleared by low-speed centrifugation.

Western Analysis:

Western analysis for the indicated signaling factors and GAPDH(endogenous control) were conducted as described in Sajan and Farese.2012 Diabetologia. 55:1446-1457, Standaert et al. 2004. J. Biol. Chem.279:24929-24934, Sajan et al. 2012. Metabolism. 61:459-469, Sajan et al.2009. Diabetologia. 52:1197-1207, Sajan et al. 2009. J. Lipid Res.50:1133-1145, and Sajan et al. 2013. Diabetologia. 56:2507-2516 usingrapid anti-phospho-Ser⁴⁷³-Akt antiserum, rabbitanti-glyceraldehyde-phosphate dehydrogenase (GAPDH) antiserum, rabbitanti-WD40-ProF antiserum, and rabbit anti-aPKC antiserum (Santa CruzBiotechnologies, Santa Cruz, Calif.); rabbit anti-phospho-Thr560/555PKC-qIA/i antiserum (Invitrogen, Carlsbad, Calif.); rabbitanti-p-Ser²⁵⁶-FoxO1 and anit-FoxO1 antiserum (Abnova, Walnut, Calif.);mouse monoclonal anti-PKC-λ/ι antibodies (Transduction Antibodies,Bedford, Mass.); and rabbit anti-phospho-Ser⁹-GSK3β antiserum, rabbitanti-phosphoSer²²⁴⁸-mTOR antiserum, and mouse anti-Akt antibodies (CellSignaling Technologies, Danvers, Mass.). Samples from experimentalgroups were compared on the same blots and corrected for recovery asneeded by measurement of GAPDH immunoreactivity.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

FIG. 7 demonstrates the effects of HFF diet and ICAP (I) or ACPD (A) onhepatic resting/basal and insulin stimulated phosphorylation ofSer⁴⁷³-Akt (FIG. 7A), Ser9-GSK3β (FIG. 7B), Thr⁵⁵⁵¹⁵⁶⁰-PKC-λ/ζ, (FIG.7C), and Thr²⁴⁴⁸-mTOR (FIG. 7D). In control low-fat-fed mice, insulinprovoked rapid increases in the activity of both aPKC (FIG. 7C) and Akt2(FIG. 7A) in the liver. In HFF mice, basal/resting andinsulin-stimulated activities of aPKC (FIG. 7C) and Akt2 (FIG. 7A) wereelevated. This is possibly due to hyperinsulinemia.

Further, in HFF mice basal/resting and insulin-stimulated activities ofaPKC (FIG. 7C) and Akt2 (FIG. 7A) were increased after acute insulintreatment to levels comparable with those of low fat fed mice.

ACPD and ICAP reduced basal/resting and exogenous insulin-stimulatedhepatic aPKC activities to levels observed in control low fat fed mice(FIG. 7C). In contract, ACPD and ICAP treatment did not alter restingAkt2 activity in livers of HFF mice, but increased insulin-stimulatedAkt2 activity therein (FIG. 7A).

Example 8 Effect of High Fat Feeding and ICAP or ACPD on Expression andPhosphorylation of Lipogenic and Gluconeogenic Enzymes, Recovery ofaPKC, FoxO1, and WD40/ProF in WD40/ProF Immunopercipitates, and Recoveryof aPKC and Akt2 Activity in Liver

Methods

Standard (C57Bl/6/SV129) mice consuming standard low-fat mouse chow wereinjected subcutaneously (s.c.) with a daily single injection of ICAP (1mg/kg body weight) or ACPD (10 mg/kg body weight). After 10 weeks offeeding a low fat diet (10% calories from fat) (control mice) or a highfat diet (40% of calories from milk fat) and daily treatment with orwithout aPKC inhibitor (ACPD or 1H-imidazole-4-carboxamide,5-amino-1-[2,3-dihydroxy-4-[(phosphono-oxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)(ICAP)), mice were injected with or without insulin (1 U/kg body weightgiven intraperitoneally) and killed 15 minutes post injection. Postmortem, liver tissue was removed and examined for immunoreactivity ofthe indicated signaling factors.

For the results shown in FIG. 8, liver tissue was prepared and mRNAlevels of SREVP-1c, FAS, PEPCK, and G6PAse were analyzed as described inExample 4.

For results shown in FIG. 9, cell lysates were prepared as described inExample 7. Nuclear preparations were also prepared as described in Sajanet al. 2009. Diabetologia. 52:1197-1207. Western analysis was conductedto evaluate the indicated immunoreactive proteins as in Example 7,except in this Example, rabbit polyclonal anti-GAPDH antiserum, rabbitpolyclonal anti-FAS antiserum, rabbit polyclonal anti-PEPCK antiserum,rabbit polyclonal anti-G6Pase antiserum (Santa Cruz Biotechnologies,Santa Cruz, Calif.), and mouse monoclonal anti-SREBP-1 antibodies (LabVision Corp., Freemont, Calif.) were used to detect the indicatedproteins.

For the results shown in FIG. 10A, liver cell lysates were prepared aspreviously described. Phosphorylated FoxO1 and un-phosphorylated FoxO1were analyzed using western blotting as previously described exceptrabbit anti-phospho-Ser²⁵⁶-FoxO1 antisera and anti-FoxO1 antiserum wasused.

For the results shown in FIGS. 10B, 10C, and 10D, immunopercipitateswere prepared from liver cell lysates that were prepared as previouslydescribed using an anti-WD40/ProF antibody. For the results in FIG. 10B,the indicated immunoreactive proteins were analyzed in theimmunopercipitates via western blot as previously described except thatrabbit anti-aPKC-λ/ι, FoxO1, or WD40/ProF antisera were used. For theresults in FIGS. 10C and 10D, activities of Akt2 (FIG. 10C) and aPKC(10D) were evaluated in the immunopercipitates as described in Example3.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

FIG. 8 demonstrates the effects of a HFF diet and ICAP (I) or ACPD (A)treatment on mRNA levels of lipogenic enzymes SREBP-1c (FIG. 8A) and FAS(FIG. 8B), as well as mRNA levels of gluconeogenic enzymes PEPCK (FIG.8C) and G6Pase (FIG. 8D) in the livers of ad libitum-fed mice. As shownin FIG. 8, HFF stimulated increases in hepatic mRNA levels of lipogenicenzymes sterol receptor element-binding protein-1c (SREBP-1c) (FIG. 8A)and fatty acid synthase (FAS) (FIG. 8C), as measured in the fed state.HFF also stimulated increases in hepatic mRNA levels of gluconeogenicenzymes PEPCK (FIG. 8B) and glucose-6-phosphatase (G6Pase) (FIG. 8D), asmeasured in the fed state. The increase in mRNA levels for each enzymecorrelated to increases in protein levels as shown in FIG. 9.

However, mRNA (FIG. 8) and protein (FIG. 9) levels of SREPB-1c, FAS,PEPCK, and G6Pase in HFF mice were not significantly different fromcontrol/low-fat fed mice after ICAP or ACPD treatment and reduction ofhepatic aPKC activity. Consonant with the idea that diminished hepaticaPKC activity was responsible for improvements in expression oflipogenic and gluconeogenic enzymes in HFF mice treated with ICAP orACPD, aPKC plays a significant role in 1) feeding- and insulin-dependentincreases in activity and expression of SREBP-1c, which is implicated inthe increase in expression of multiple lipogenic enzymes; and 2)fasting-dependent increases in expression of PEPCK and G6Pase.

In livers of T2DM humans, and rodents, Akt activity/activation isdiminished and expression of PEPCK/G6Pase is increased. However, in theHFF mice in this Example, the presence of increased PEPCK/G6Paseexpression in the face of elevated hepatic Akt2 activity seemed at odds.This conundrum was resolved by finding that Akt2-dependentphosphorylation of Ser256-FoxO1, which by its phosphorylation andnuclear exclusion mediates insulin-dependent decreases in gluconeogenicenzyme expression, was markedly diminished basally and almost completelyunresponsive to exogenous insulin treatment in livers of HFF mice (FIG.10A).

In contrast to FoxO1 phosphorylation but in keeping with increases inhepatic Akt2 activity in HFF mice, Akt-dependent phosphorylation of bothSer⁹-glycogensynthase kinase (GSK)-3β, which, by inhibiting GSK3β,mediates stimulatory effects on glycogen synthesis, and Ser²⁴⁴⁸-mTOR,which, by activating S6 kinase, mediates stimulatory effects onlipogenesis, was increased by high fat feeding, as well as by exogenousinsulin treatment. Moreover, this was not altered by ICAP or ACPDtreatment (FIGS. 7B and 7D, Example 7). Accordingly, the defect in FoxO1phosphorylation in HFF mice was relatively specific and did not reflecta generalized defect in hepatic Akt-dependent phosphorylation.

Treatment of HFF mice with ICAP or ACPD fully or partially restoredbasal/resting and insulin-stimulated hepatic FoxO1 phosphorylation (FIG.10A). This restoration suggested that activation of hepatic aPKCcontributed to the impairment of FoxO1 phosphorylation in HFF liver.

Moreover, the stimulatory effect of ICAP or ACPD on FoxO1phosphorylation provided a reasonable explanation for theimprovement/suppression of gluconeogenic enzyme expression in HFF mice(FIG. 8).

Findings that FoxO1 phosphorylation was diminished despite heightenedAkt2 activity, and was increased in basal/resting conditions by ICAP orACPD without change in overall cellular Akt2 activity, suggested thatAkt-dependent FoxO1 phosphorylation and inhibition thereof byHFF-activated aPKC might be compartmentalized. In adipocytes, thescaffold protein WD40/ProF binds FoxO1 and activated forms of both Aktand aPKC and, moreover, is needed for Akt-mediated phosphorylation ofFoxO1 but not other Akt substrates, such as GSK3β and mTORC1, i.e., apattern of selective inhibition of Akt substrates identical to thatobserved above in HFF liver. Indeed, as shown in FIG. 10, liver levelsand activity of aPKC recovered in WD40/ProF immunoprecipitates wereincreased, particularly by HFF and to a lesser degree by exogenousinsulin treatment (FIGS. 10B and 10D).

Activity of Akt recovered in WD40/ProF immunoprecipitates was increasedby insulin in control/LFF mice (FIG. 10C). Basal/resting andinsulin-stimulated activities of Akt associated with WD40/ProF werediminished by HFF (FIG. 10C). Treatment of HFF mice with ICAP or ACPDdiminished aPKC binding to WD40/Prof (FIGS. 10B and 10D) butsimultaneously increased WD40/ProF-associated Akt activity (FIG. 10C)and total cellular FoxO1 phosphorylation (FIG. 10A), thereby decreasinggluconeogenic enzyme expression (FIG. 8).

Example 9 Effect of High Fat Feeding and ICAP or ACPD on GlucoseTolerance, Serum Levels of Glucose, Insulin, Triglycerides, andCholesterol, Body Weight, Food Intake, Liver Triglycerides, AbdominalFat Deposits, and Hepatic Fat

Methods

Standard (C57Bl/6/SV129) mice consuming standard low-fat mouse chow wereinjected subcutaneously (s.c.) with a daily single injection with orwithout either ICAP (1 mg/kg body weight) or ACPD (10 mg/kg bodyweight)). After 9 weeks of treatments, mice were subjected to glucosetolerance testing using methods generally known in the art. Briefly, 2mg glucose/kg body weight was injected intraperitoneally. After theglucose injection, blood glucose levels were measured at 0, 30, 60, 90and 120 minutes post-injection. 15 minutes prior to killing, mice wereinjected with or without insulin (1 U/kg body weight). Serum glucose,triglycerides, and serum cholesterol were measured using methodsgenerally known in the art. Liver triglycerides were also measured usingmethods generally known in the art. Over the 10 weeks, body weight andfood intake were measured weekly. Overall change in bodyweight was alsoevaluated. Additionally, epididymal plus retroperitoneal fat (alsoabbreviated PG/PN fat) was measured and evaluated as g/100 g of bodyweight. After killing, liver tissue samples were sectioned and preparedfor Oil red O staining by methods generally known in the art. Sampleswere Oil Red O stained to observe hepatic fat contents.

Where appropriate, data are expressed as mean±SEM, and P values weredetermined by one-way ANOVA and least-significant multiple comparisonmethods.

Results

Treatment with ICAP or ACPD improved glucose tolerance, basal glucoselevels in fasting and fed conditions, and acute effects of insulin onserum glucose levels in HFF to levels indistinguishable from those seenin control chow-fed mice (FIG. 11). On the other hand, although fastinginsulin levels in HFF mice were improved by ICAP or ACPD treatment,serum insulin levels were still modestly elevated (FIG. 11C). It ispossible that the remaining hyperinsulinemia in ICAP- and ACPD-treatedHFF mice was sufficient to compensate for any residual insulinresistance.

In addition to normalization or near normalization of glucosehomeostasis, treatment with ICAP and ACPD largely prevented (a) weightgain without altering food intake (FIGS. 12A, 12B and 12E); (b)increases in liver triglycerides (FIG. 12C) and fat content (FIG. 13);and (c) increases in serum triglycerides and cholesterol levels (FIGS.11D and 11E). In addition, HFF-induced increases in abdominal fat depotswere improved by ICAP and trended downward with ACPD treatment (FIG.12). Remaining abnormalities in lipid homeostasis may reflect effects ofresidual hyperinsulinemia on lipid synthesis in adipocytes or remainingsmall increases in lipogenic enzymes in ICAP- and ACPD-treated HFF mice.

Example 10 Effect of Ceramide on Hepatic aPKC Activity and the Effect ofHFF Diet on Hepatic Levels of Ceramide and Sphingomyelin Species

Methods

Standard (C57Bl/6/SV129) mice consumed a standard low-fat or high-fatdiet for 10 weeks. At 10 weeks mice were treated with or without insulinfor 15 min prior to killing. Liver cell lysates were prepared as inExample 5 and aPKC activity assay was performed on the aPKCimmunoprecipitate as in Example 5, except here the activity assays werecarried out in the presence of the indicated concentrations of ceramide.Additionally, the levels of ceramide and sphingomyelin species in lipidextracts prepared from the liver cell lysates.

Ceramide and sphingomyelin species were measured by liquidchromatography tandem mass spectrometry analysis of the lipid extractsof the liver lysates.

Where appropriate, data are expressed as mean±SEM, and P values weredetermined by one-way ANOVA and least-significant multiple comparisonmethods.

Results

As ceramide provokes hepatic abnormalities in HFF mice and directlyactivates aPKC it was tested whether ceramide contributed to increasesin aPKC activity in the HFF mice. Ceramide strongly activated aPKC whenadded to immunoprecipitates prepared from liver lysates of LFF/controlmice but had only a weak effect on aPKC immunoprecipitated from lysatesof HFF mice and diminished activity of aPKC immunoprecipitated fromlysates of HFF mice treated acutely with insulin (FIG. 14). (Note:ceramide has biphasic effects on aPKC activity, with inhibition afterstimulation in dose response studies) Similarly, PIP3 inhibits aPKCactivity when added in excess). On the other hand, acute insulintreatment, possibly acting via PIP3, elicited further increases in aPKCactivity in HFF mice (FIG. 14 [compare with FIG. 9A]). Ceramide levelswere increased in HFF mice as shown in FIG. 14.

Example 11 Effect of ACPD in Lean or Ob/Ob Mice on Glucose Tolerance andPhosphorylation of aPKC and Akt

Methods

Male mice, 3-5 months of age, were used throughout these studies. Theob/ob mice and their lean counterparts (ob⁺) were purchased from JacksonLaboratories (Bar Harbor, Me., USA) at 2-3 months of age and studiedover a 10-week period. During the 3rd to 5th month, they were injectedsubcutaneously once daily with ACPD (10 mg/kg body weight) inphysiological saline vehicle or vehicle alone. During the 9th week,glucose tolerance was measured after an overnight fast byintraperitoneal injection of 2 mg glucose per kg body weight, asdescribed in Example 9. At the 10th week, mice were injected with orwithout insulin (1 U/kg body weight) 15 minutes prior to killing andrapid harvesting of tissues.

All experimental procedures involving animals were approved by theInstitutional Animal Care and Use Committees of the University of SouthFlorida College of Medicine, and the James A. Haley VeteransAdministration Medical Center Research and Development Committee, Tampa,Fla.

Tissue lysates were prepared and western blotting to detectimmunoreactive p-ser473-Akt and p-thr555/560-PKC-ι/λ/4 was performed aspreviously described in the Examples herein. Where appropriate, data areexpressed as mean±SEM, and P values were determined by one-way ANOVA andleast-significant multiple comparison methods.

Results

Treatment of ob/ob mice with ACPD (10 mg/kg body weight given once dailysubcutaneously for 10 weeks) led to partially decreased phosphorylationof hepatic aPKC in both resting (but stimulated by endogenoushyperinsulinaemia-See Example 12) and exogenous-insulin-stimulatedconditions (FIG. 16A). In contrast to aPKC, the increases in Aktphosphorylation observed in livers of ob/ob mice were not altered byACPD treatment (FIG. 16C). Additionally, the impairmentinsulin-stimulated phosphorylation of aPKC was improved in muscles ofob/ob mice following ACPD treatment (FIG. 16B), and Akt phosphorylationtended to increase (FIG. 16D).

As seen in FIG. 17, although fasting blood glucose levels were onlymildly increased in ob/ob mice relative to lean control mice, glucosetolerance, as measured in the peritoneal glucose tolerance test (GTT),was more strikingly diminished. Treatment of ob/ob mice with ACPD led toimprovements in both fasting blood glucose levels and GTT-determinedglucose tolerance (FIG. 17A). On the other hand, the elevated fastingand post-glucose insulin levels seen in ob/ob mice during the GTT (FIG.17B), showed little or no change.

Example 12 Effect of ACPD on Food Intake, Body Weight, Fat Deposition,Serum Triglycerides, and Liver Triglycerides in Ob/Ob mice

Methods

Male mice, 3-5 months of age, were used throughout these studies. Theob/ob mice and their lean counterparts (ob⁺) were purchased from JacksonLaboratories (Bar Harbor, Me., USA) at 2-3 months of age and studiedover a 10-week period. During the 3rd to 5th month, they were injectedsubcutaneously once daily with ACPD (10 mg/kg body weight) inphysiological saline vehicle or vehicle alone. During the 9th week,glucose tolerance was measured after an overnight fast byintraperitoneal injection of 2 mg glucose per kg body weight, asdescribed in Example 9. At the 10th week, mice were injected with orwithout insulin (1 U/kg body weight) 15 minutes prior to killing andrapid harvesting of tissues. Body weight and food intake was measuredweekly. The combined weight of epididymal plus retroperitoneal fatdepots (abdominal fat depots) was measured. Serum and livertriglycerides were obtained and measured as previously described in theExamples herein.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

All experimental procedures involving animals were approved by theInstitutional Animal Care and Use Committees of the University of SouthFlorida College of Medicine, and the James A. Haley VeteransAdministration Medical Center Research and Development Committee, Tampa,Fla.

Results Food intake was increased in ob/ob mice and ACPD treatment hadlittle or no effect on food intake as shown in FIG. 18B. On the otherhand, increases in body weight during the 10-wek study period trendeddownward in ob/ob mice treated with ACPD (FIG. 18A). Further, in ob/obmice, the combined weights of epididymal plus retroperitoneal fat depots(FIG. 18C), serum triglycerides (FIG. 18D), and liver triglycerides(FIG. 18E) diminished following ACPD treatment.

Example 13 Effects of ACPD on Phosphorylation/Activities of pSer⁴⁷³-Aktand p-Thr⁵⁵⁶⁵⁶⁰-PKC-λ/ι) in Resting/Basal and Insulin-StimulatedConditions and During Treatment with aPKC Inhibitor in Liver and MuscleLysates of Lean Ob⁺ and Obese-Phase Ob/Ob Mice

Methods

Male mice, 3-5 months of age, were used throughout these studies. Theob/ob mice and their lean counterparts (ob⁺) were purchased from JacksonLaboratories (Bar Harbor, Me., USA) at 2-3 months of age and studiedover a 10-week period. During the 3rd to 5th month, they were injectedsubcutaneously once daily with ACPD (10 mg/kg body weight) inphysiological saline vehicle or vehicle alone. During the 9th week,glucose tolerance was measured after an overnight fast byintraperitoneal injection of 2 mg glucose per kg body weight, asdescribed in Example 9. At the 10th week, mice were injected with orwithout insulin (1 U/kg body weight) 15 minutes prior to killing andrapid harvesting of tissues. Body weight and food intake was measuredweekly. ACPD in all of the Examples disclosed herein does not inhibitkinases, Akt2, FGFR1/2/3/4, mTOR, GSK3β, IRAK1/4, JAK1/2, MEK1, ERK1/2,JNK 1/2, PKA, Src, ROCK 2, ROS1, or PI3Kα/α as tested by LifeTechnologies (Madison Wis., USA)

Liver and muscle cell lysates were prepared as described as in Example5. Western analyses were conducted as described in these Example 7except using: rabbit polyclonal anti-phospho-serine-473-Akt,anti-glyceraldehyde-phosphate dehydrogenase (GAPDH), anti-WD40/ProF,anti-aPKC antisera, anti-FAS, ant-G6Pase, anti-PEPCK, anti-p65/RelA/NFkBantisera (Santa Cruz Biotechnologies, Santa Cruz, Calif., USA); rabbitpolyclonal anti-phospho-threonine-560/555-PKC-ι-λ antiserum (Invitrogen,Carlsbad, Calif., USA); rabbit polyclonal anti-p-serine-256-FoxO1 andanti-FoxO1 antisera (Abnova, Walnut, Calif., USA); mouse monoclonalanti-PKC-λ/ι antibodies (Transduction Antibodies, Bedford, Mass., USA);rabbit polyclonal anti-phospho-serine-9-GSK3β, anti-ACC andanti-phospho-serine-2248-mTOR antisera, and mouse monoclonal anti-Aktantibodies (Cell Signaling Technologies, Danvers, Mass., USA); mousemonoclonal anti-SREBP-1 antibodies (Lab Vision Corp., Freemont, Calif.,USA); horseradish-peroxidase-conjugated goat anti-mouse and anti-rabbitsecondary antibodies (Biorad, Hercules, Calif., USA); andhorseradish-peroxidase-conjugated AffiniPure donkey anti-mouse andanti-rabbit secondary antibodies (Jackson ImmunoResearch Labs, WestGrove, Pa., USA). Samples from experimental groups were compared on thesame blots and routinely checked with loading controls.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

Results are shown in FIG. 19. In lean control ob⁺ mice, insulinincreased phosphorylation of both aPKC and Akt in liver and muscle(FIGS. 19A, 19B,19C, 19D). However, in muscles of ob/ob mice,resting/basal and insulin-stimulated increases in phosphorylation ofaPKC were significantly diminished. Insulin-stimulated Aktphosphorylation trended downward, relative to findings in lean ob⁺ mice(FIGS. 19B and 19D).

In contrast to muscle, phosphorylation of hepatic aPKC in ob/ob mice wasincreased in resting/basal conditions to a level comparable to if notgreater than that seen with insulin treatment in lean ob⁺ mice, andacute exogenous insulin treatment had no further effect on aPKCphosphorylation (FIG. 19C), suggesting that hepatic aPKC was maximallyactivated in obese-phase ob/ob mice by endogenous hyperinsulinemia (seebelow) and/or other factors. Partly similar to hepatic aPKC,resting/basal hepatic Akt phosphorylation was elevated in ob/ob mice,and with acute exogenous insulin treatment, Akt phosphorylation waselevated to levels not significantly different from those seen in leanob⁺ mice (FIG. 19A).

Treatment of ob/ob mice with ACPD reduced the elevated resting/basallevels of hepatic aPKC phosphorylation almost to normal andsubstantially diminished stimulatory effects of exogenous insulintreatment on hepatic aPKC phosphorylation (FIG. 19C). In contrast toaPKC, hepatic Akt phosphorylation in ob/ob mice was not alteredsignificantly by ACPD treatment (FIG. 19A). Impairments ininsulin-stimulated phosphorylation of both aPKC and Akt in muscles ofob/ob mice were significantly improved or trended upward with ACPDtreatment (FIGS. 19B and 19D).

Example 14 Phosphorylation of Akt Substrates and Association of aPKCwith Hepatic WD40/Prof in Resting/Basal and Insulin-StimulatedConditions, and During Treatment with aPKC Inhibitor, ACPD, in Livers ofLean Ob+ and Obese-Phase Ob/Ob Mice

Methods

Male mice, 3-5 months of age, were used throughout these studies. Theob/ob mice and their lean counterparts (ob⁺) were purchased from JacksonLaboratories (Bar Harbor, Me., USA) at 2-3 months of age and studiedover a 10-week period. During the 3rd to 5th month, they were injectedsubcutaneously once daily with ACPD (10 mg/kg body weight) inphysiological saline vehicle or vehicle alone. During the 9th week,glucose tolerance was measured after an overnight fast byintraperitoneal injection of 2 mg glucose per kg body weight, asdescribed in Example 9. At the 10th week, mice were injected with orwithout insulin (1 U/kg body weight) 15 minutes prior to killing andrapid harvesting of tissues. Body weight and food intake was measuredweekly. ACPD in all of the examples disclosed herein does not inhibitkinases, Akt2, FGFR1/2/3/4, mTor, GSK3β, IRAK1/4, JAK1/2, MEK1, ERK1/2,JNK 1/2, PKA, Src, ROCK 2, ROS1, or PI3Kα/α as tested by LifeTechnologies (Madison Wis., USA)

Liver cell lysates were prepared as described as in Example 5. Westernanalyses were conducted as described in Example 7 except using: rabbitpolyclonal anti-phospho-serine-473-Akt, anti-glyceraldehyde-phosphatedehydrogenase (GAPDH), anti-WD40/ProF, anti-aPKC antisera, anti-FAS,ant-G6Pase, anti-PEPCK, anti-p65/RelA/NFkB antisera (Santa CruzBiotechnologies, Santa Cruz, Calif., USA); rabbit polyclonalanti-phospho-threonine-560/555-PKC-ι-λ antiserum (Invitrogen, Carlsbad,Calif., USA); rabbit polyclonal anti-p-serine-256-FoxO1 and anti-FoxO1antisera (Abnova, Walnut, Calif., USA); mouse monoclonal anti-PKC-λ/ιantibodies (Transduction Antibodies, Bedford, Mass., USA); rabbitpolyclonal anti-phospho-serine-9-GSK3β, anti-ACC andanti-phospho-serine-2248-mTOR antisera, and mouse monoclonal anti-Aktantibodies (Cell Signaling Technologies, Danvers, Mass., USA); mousemonoclonal anti-SREBP-1 antibodies (Lab Vision Corp., Freemont, Calif.,USA); horseradish-peroxidase-conjugated goat anti-mouse and anti-rabbitsecondary antibodies (Biorad, Hercules, Calif., USA); andhorseradish-peroxidase-conjugated AffiniPure donkey anti-mouse andanti-rabbit secondary antibodies (Jackson ImmunoResearch Labs, WestGrove, Pa., USA). Samples from experimental groups were compared on thesame blots, and routinely checked with loading controls.

To determine association of aPKC and Akt, aPKC and Akt immunoreactiveprotein was evaluated in WD40/ProF immunopercipitates as previouslydescribed in Example 8.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

In lean ob⁺ mice, in conjunction with increases in Akt phosphorylation,insulin increased hepatic FoxO1 phosphorylation (FIG. 20A). In ob/obmice, however, despite hyperinsulinemia and elevated levels of hepaticAkt phosphorylation in resting/basal and insulin-stimulated conditions,FoxO1 phosphorylation remained at a lower or modestly diminishedresting/basal level, and, moreover, responded poorly if at all to acuteinsulin treatment (FIG. 20A). Yet, with ACPD treatment of ob/ob mice,both resting/basal and insulin-stimulated FoxO1 phosphorylationincreased to levels comparable to those seen in insulin-stimulated leanob⁺ mice (FIG. 20A).

In contrast to FoxO1, phosphorylation of both mTOR and GSK3β wasincreased, not only by insulin in lean ob⁺ mice, but also inresting/basal conditions (presumably this reflects hyperinsulinemia andincreased Akt activation), and following exogenous insulin treatment inob/ob mice (FIGS. 20B and 20C). Moreover, ACPD treatment did notsubstantially alter the elevations in mTOR and GSK3β phosphorylationseen in ob/ob mice (FIGS. 20B and 20C).

Insulin acutely increased recovery of aPKC and Akt in WD40/ProFimmunoprecipitates (FIGS. 20D and 20E). However, in ob/ob mice theresting/basal level of aPKC associated with WD40/ProF was increasedmaximally, as it did not increase further with insulin treatment (FIG.20D). In contrast, Akt associated with WD40/ProF was diminished basallyand increased poorly following insulin treatment (FIG. 20E). Further,ACPD treatment diminished aPKC association with WD40/ProF (FIG. 20D).This was accompanied by increased association of Akt with WD40/ProF(FIG. 20E).

Example 15 Effects of ACPD on mRNA Levels of Hepatic SREBP-1c, FAS, ACC,TNF-α, PEPCK, G6PASE, Hepatic Nuclear Levels of the Proteolytic Fragmentof SREBP-1c and Active Subunit of NFκB, and Hepatic Lysate Protein Levelof FAS, ACC, PEPCK and G6Pase in Ob/Ob Mice

Methods

Male mice, 3-5 months of age, were used throughout these studies. Theob/ob mice and their lean counterparts (ob⁺) were purchased from JacksonLaboratories (Bar Harbor, Me., USA), at 2-3 months of age and studiedover a 10-week period. During the 3rd to 5th month, they were injectedsubcutaneously once daily with ACPD (10 mg/kg body weight) inphysiological saline vehicle or vehicle alone. During the 9th week,glucose tolerance was measured after an overnight fast byintraperitoneal injection of 2 mg glucose per kg body weight, asdescribed in Example 9. At the 10th week, mice were injected with orwithout insulin (1 U/kg body weight) 15 minutes prior to killing andrapid harvesting of tissues. mRNA Quantification. Tissues and RNA wasprepared as described in Example 4.

Quantification of mRNA was performed using quantitative PCR as describedin Example 4. Primers, in addition to those used in Example 4 are asfollows: TNF-α Forward SEQ ID NO: 11 ACGGCATGGATCTCAAAGAC, TNF-α ReverseSEQ ID NO: 12 AGATAGCAAATCGGCTGACG ACC Forward SEQ ID NO: 13GACTTCATGAATTTGCTGAT and ACC Reverse SEQ ID NO 14: AAGCTGAAAGCTTTCTGTCT.

Nuclear preparations were prepared as previously described in Example 6.Western analysis was performed as described in Example 14.

Data are expressed as mean±SEM, and P values were determined by one-wayANOVA and least-significant multiple comparison methods.

Results

Results are shown in FIGS. 21, 22, and 23. In ob/ob mice, hepaticexpression of gluconeogenic enzymes, PEPCK (FIG. 21E) and G6Pase (FIG.21F), lipogenic enzymes, SREBP-1c (FIG. 21A), FAS (FIG. 21B) and ACC(FIG. 21C), and proinflammatory tumor necrosis factor-α (TNF-α) (FIG.21D) was elevated, but virtually restored to normal with ACPD treatment(FIG. 21). Similarly, in ob/ob mice, nuclear protein levels of theactive fragment of SREBP-1c (FIG. 22A) and nuclear protein levels of theactive p65/RelA subunit of NFκB (FIG. 22B) were increased (FIG. 22), andlysate protein levels of ACC (FIG. 23A), FAS (FIG. 23B), PEPCK (FIG.23C) and G6Pase (FIG. 23D) were increased, and ACPD treatment largelyreversed these increases (FIG. 23).

Example 16 Effect of Insulin Stimulation on aPKC and Akt2 Activity inthe Brains of High Fat Fed and Ob/Ob Mice INTRODUCTION

Insulin-resistant and (glucose intolerant) syndromes, including obesityand metabolic syndrome, and Alzheimer's disease (AD) are endemic closelyrelated disorders. Insulin resistance predisposes a subject to ADdevelopment. In Western/Westernized populations, insulin-resistantobesity and the metabolic syndrome (O/MetSYN) are present in 34% of theadult population, and frequently progress to type 2 diabetes mellitus(T2DM), which is present in 27% of people over the age of 65 (CDC data,2011). Moreover, Alzheimer's disease (AD) is estimated to afflict 13% ofpeople over the age of 65 and 45% of people over the age of 85.Furthermore, AD is 50% more prevalent in T2DM, and T2DM prevalence isapproximately 2-fold greater in AD. As O/MetSyn/T2DM generally precedesAD, it is suspected that O/MetSyn/T2DM predisposes to the development ofAD.

In this Example, the effect of insulin stimulation on aPKC and Akt2activity in the brains of high fat fed (HFF) and ob/ob mice wasevaluated. HFF and ob/ob mice are insulin-resistant, hyperinsulinemic,and develop obesity, metabolic syndrome, and T2DM. Thus, these mice area model of a subject predisposed to AD.

Methods

Standard lab mice were fed a high fat diet in which 40% of calories werederived from milk fat or a standard chow containing 10% fat (“control”or “con” mice) for 10 weeks. Standard lab mice (“control” or “con”) andob/ob (OB) mice were fed the standard chow containing 10% fat. At 10weeks some mice in each group were administered 1 U/kg insulinintraperitoneally 15 minutes prior to killing. Brain tissue was removed.Preparations containing brain proteins from the brain tissue samples wasanalyzed for aPKC and Akt activities via Western Blot followed byquantitative scanning of the blots.

Results

Results from this experiment are demonstrated in FIG. 25. Treatment ofcontrol mice intraperiotneally with a maximal dose of insulin leads toincreases in brain activities and phosphorylation of both Akt (FIGS. 25Band 25D) and aPKC (FIGS. 25A and 25C). It has been demonstrated, Akt andaPKC are activated in parallel by the product of phosphatidylinositol3-kinase (PI3K) and phosphatidytlinositol-3, 4, 5-(PO₄)₃(PIP₃) duringinsulin action. Together, these mediate most metabolic actions ofinsulin in other tissues as well. However, as demonstrated in thisExample, the basal/resting activities of both Akt and aPKC were elevatedin HFF and ob/ob mice. Moreover, there was little or no further responseof these proteins in response to exogenous insulin treatment (FIG. 25).This suggests that the hyperinsulinemia present in the HFF and ob/obmice had maximally activated both Akt and aPKC, even in basal/restingconditions.

Example 17 Effect of Insulin Stimulation on Phosphorylation of the AktSubstrates FoxO1, FoxO3a, GSK31, and mTOR INTRODUCTION

Insulin-resistant and (glucose intolerant) syndromes, including obesityand metabolic syndrome, and Alzheimer's disease (AD) are endemic closelyrelated disorders. Insulin resistance predisposes a subject to ADdevelopment. In Western/Westernized populations, insulin-resistantobesity and the metabolic syndrome (O/MetSYN) are present in 34% of theadult population, and frequently progress to type 2 diabetes mellitus(T2DM), which is present in 27% of people over the age of 65 (CDC data,2011). Moreover, Alzheimer's disease (AD) is estimated to afflict 13% ofpeople over the age of 65 and 45% of people over the age of 85.Furthermore, AD is 50% more prevalent in T2DM, and T2DM prevalence isapproximately 2-fold greater in AD. As O/MetSyn/T2DM generally precedesAD, it is suspected that O/MetSyn/T2DM predisposes to the development ofAD.

In this Example, the effect of insulin stimulation on thephosphorylation of Akt substrates FoxO1, FoxO3a, GSK3β, and mTOR in thebrains of high fat fed (HFF) and ob/ob mice was evaluated. The HFF andob/ob mice are insulin-resistant, hyperinsulinemic, and develop obesity,metabolic syndrome, and T2DM. Thus, these mice are a model of a subjectpredisposed to AD.

Methods

Standard lab mice were fed a high fat diet in which 40% of calories werederived from milk fat or a standard chow containing 10% fat (“control”or “con” mice) for 10 weeks. Standard lab mice (“control” or “con”) andob/ob (OB) mice were fed the standard chow containing 10% fat. At 10weeks some mice in each group were administered 1 U/kg insulinintraperitoneally 15 minutes prior to killing. Brain tissue was removed.Preparations containing brain proteins from the brain tissue samples wasanalyzed for aPKC and Akt activities via Western Blot usingphospho-peptide-specific antisera followed by quantitative scanning ofthe blots.

Results

The results from this experiment are demonstrated in FIGS. 26 and 27.Phosphorylation of the four substrates evaluated was elevated in thebasal/resting state of HFF and ob/ob mice (FIGS. 26 and 27) at levelscomparable to those achieved by maximal insulin treatment of controlmice (FIG. 25)). Moreover, there was little or no furtherphosphorylation of these proteins in response to exogenous insulintreatment (FIGS. 26 and 27). This suggests that the hyperinsulinemiapresent in the HFF and ob/ob mice had maximally activated both Akt andaPKC, even in basal/resting conditions.

Example 18 Effect of ICAPP on aPKC Activity in the Brains of Control andHeterozygous Muscle-Specific PKC-λ Knockout Mice INTRODUCTION

Insulin-resistant and (glucose intolerant) syndromes, including obesityand metabolic syndrome, and Alzheimer's disease (AD) are endemic closelyrelated disorders. Insulin resistance predisposes a subject to ADdevelopment. In Western/Westernized populations, insulin-resistantobesity and the metabolic syndrome (O/MetSYN) are present in 34% of theadult population, and frequently progress to type 2 diabetes mellitus(T2DM), which is present in 27% of people over the age of 65 (CDC data,2011). Moreover, Alzheimer's disease (AD) is estimated to afflict 13% ofpeople over the age of 65 and 45% of people over the age of 85.Furthermore, AD is 50% more prevalent in T2DM, and T2DM prevalence isapproximately 2-fold greater in AD. As O/MetSyn/T2DM generally precedesAD, it is suspected that O/MetSyn/T2DM predisposes to the development ofAD.

In this Example, the effect of ICAPP on aPKC activity in the brains ofcontrol and heterozygous muscle-specific PKC-λ knockout mice wasevaluated. The heterozygous knockout mice are another model of systemicinsulin resistance that stems from a specific defect in glucosetransport in the skeletal muscle of these mice. This defect, viahyperinsulinemia, activates hepatic aPKC and causes activation ofgluconeogenesic and lipogenesic pathways. (See also FIG. 24). This leadsto obesity, metabolic syndrome, and type 2 diabetes. Thus, these miceare a model of a subject predisposed to AD.

Methods

Standard lab mice (“control” or “con” mice) and heterozygousmuscle-specific PKC-λ knockout mice were fed standard chow containing10% fat. Some mice were treated for 8 days with ICAPP. Some mice wereacutely treated with insulin (1 U/kg body weight administeredintraperitoneally) 15 minutes prior to killing. Brain tissues werecollected and prepared for phospho-protein analysis. Activity/activationof aPKC was assessed by Western blot analyses of phospho-proteinimmunoreactivity followed by quantitative scanning of the blots.

Results

The results from this experiment are demonstrated in FIG. 28. In thismodel, aPKC activity in the brain was decreased. It is not believed thatICAPP itself crosses the blood brain barrier. Thus, these resultssuggest that improvement in hepatic aPKC in these knockout mice and thesubsequent restoration of relatively normal hepatic gluconeogenesis andoverall glucose homeostasis can influence aPKC activity in the brain.

1. A method of treating or preventing an aPKC abnormality in a subjectin need thereof comprising: administering an effective amount of an aPKCinhibitor or a derivative thereof to the subject, where the aPKCinhibitor has a formula according to Formula I:

where each R₁, when taken separately, is independently selected from thegroup consisting of: hydrogen, halo, C—C6 alkyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl, or, when taken together with the atomsto which they are attached, form a C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring, and where R₂ is selected from the group consistingof: hydrogen, halo, C1-C6 alkyl, tert-butyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkyl furan, C2-C6 alkenyl furan, C1-C6 alkylsulfanyl, aryl,heteroaryl, aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy,heteroaralkoxy, nitro, cyano, amino, C1-C6 alkylamino, di-(C1-C6alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, (C1-C6 alkoxy)carbonyl,(C1-C6 alkyl)aminocarbonyl, di-(C1-C6 alkyl)aminocarbonyl, arylcarbonyl,aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, arylsulfonyl, and2-acetyl-3-oxobutanimidoyl cyanide.
 2. The method of claim 1, whereinthe derivative is a salt or an alcohol of Formula I.
 3. The method ofclaim 1, wherein R₂ is selected from the group consisting of:2-propylfuran, (E)-2-(prop-1-en-1-yl)furan, and tert-butyl.
 4. Themethod of claim 1, wherein the R₁ together with the atoms to which theyare attached form benzene.
 5. The method of claim 1, wherein theeffective amount ranges from about 0.001 mg to about 1,000 mg.
 6. Themethod of claim 1, wherein the effective amount is administered in adosage form formulated for oral, vaginal, intravenous, transdermal,subcutaneous, intraperitoneal, or intramuscular administration.
 7. Themethod of claim 1, wherein the aPKC abnormality is selected from thegroup consisting of obesity, glucose intolerance, metabolic syndrome,hyperinsulinemia, hepatosteatosis, non-alcoholic cirrhosis,hypertriglyceridemia, hypercholesterolemia, polycystic ovary disease,and Alzheimer's disease.
 8. A method for treating or preventing an aPKCabnormality comprising: contacting a hepatic cell with an effectiveamount of an aPKC inhibitor or a derivative thereof, where the aPKCinhibitor has a formula according to Formula I:

where each R₁, when taken separately, is independently selected from thegroup consisting of: hydrogen, halo, C—C6 alkyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl, or, when taken together with the atomsto which they are attached, form a C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring, and where R₂ is selected from the group consistingof: hydrogen, halo, C1-C6 alkyl, tert-butyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkyl furan, C2-C6 alkenyl furan, C1-C6 alkylsulfanyl, aryl,heteroaryl, aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy,heteroaralkoxy, nitro, cyano, amino, C1-C6 alkylamino, di-(C1-C6alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, (C1-C6 alkoxy)carbonyl,(C1-C6 alkyl)aminocarbonyl, di-(C1-C6 alkyl)aminocarbonyl, arylcarbonyl,aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, arylsulfonyl, and2-acetyl-3-oxobutanimidoyl cyanide.
 9. The method of claim 8, thederivative is a salt or an alcohol of Formula I.
 10. The method of claim8, wherein the effective amount ranges from about 0.001 mg to about 100mg. 11.-14. (canceled)
 15. A pharmaceutical formulation for treating orpreventing an aPKC abnormality in a subject in need thereof comprising:an effective amount of aPKC inhibitor or a derivative; and apharmaceutically acceptable carrier, where the aPKC inhibitor has aformula according to Formula I:

where each R₁, when taken separately, is independently selected from thegroup consisting of: hydrogen, halo, C—C6 alkyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkylsulfanyl, aryl, heteroaryl, aryloxy, heteroaryloxy, aralkyl,heteroaralkyl, aralkoxy, heteroaralkoxy, nitro, cyano, amino, C1-C6alkylamino, di-(C1-C6 alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl,(C1-C6 alkoxy)carbonyl, (C1-C6 alkyl)aminocarbonyl, di-(C1-C6alkyl)aminocarbonyl, arylcarbonyl, aryloxycarbonyl, (C1-C6alkyl)sulfonyl, and arylsulfonyl, or, when taken together with the atomsto which they are attached, form a C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring, and where R₂ is selected from the group consistingof: hydrogen, halo, C1-C6 alkyl, tert-butyl, C2-C6 alkenyl, C1-C6haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, C2-C6 alkynyl, C3-C8cycloalkenyl, (C3-C8 cycloalkyl)C1-C6 alkyl, (C3-C8 cycloalkyl)C2-C6alkenyl, (C3-C8 cycloalkyl) C1-C6 alkoxy, C3-C7 heterocycloalkyl, (C3-C7heterocycloalkyl)C1-C6 alkyl, (C3-C7 heterocycloalkyl)C2-C6 alkenyl,(C3-C7 heterocycloalkyl)C1-C6 alkoxyl, hydroxy, carboxy, oxo, sulfanyl,C1-C6 alkyl furan, C2-C6 alkenyl furan, C1-C6 alkylsulfanyl, aryl,heteroaryl, aryloxy, heteroaryloxy, aralkyl, heteroaralkyl, aralkoxy,heteroaralkoxy, nitro, cyano, amino, C1-C6 alkylamino, di-(C1-C6alkyl)amino, carbamoyl, (C1-C6 alkyl)carbonyl, (C1-C6 alkoxy)carbonyl,(C1-C6 alkyl)aminocarbonyl, di-(C1-C6 alkyl)aminocarbonyl, arylcarbonyl,aryloxycarbonyl, (C1-C6 alkyl)sulfonyl, arylsulfonyl, and2-acetyl-3-oxobutanimidoyl cyanide.
 16. The pharmaceutical formulationof claim 16, wherein the derivative is a salt or an alcohol of FormulaI.
 17. The pharmaceutical formulation of claim 15, wherein R₂ isselected from the group consisting of: 2-propylfuran,(E)-2-(prop-1-en-1-yl)furan, and tert-butyl.
 18. The pharmaceuticalformulation as in claim 15, wherein the R₁ together with the atoms towhich they are attached form benzene.
 19. The pharmaceutical formulationas in claim 15, wherein the effective amount ranges from about 0.001 mgto about 1,000 mg.
 20. The pharmaceutical formulations as in claim 15,wherein the aPKC abnormality is selected from the group consisting ofobesity, glucose intolerance, metabolic syndrome, hyperinsulinemia,hepatosteatosis, non-alcoholic cirrhosis, hypertriglyceridemia,hypercholesterolemia, polycystic ovary disease, and Alzheimer's disease.